Delamination resistant pharmaceutical glass containers containing active pharmaceutical ingredients

ABSTRACT

The present invention is based, at least in part, on the identification of a pharmaceutical container formed, at least in part, of a glass composition which exhibits a reduced propensity to delaminate, i.e., a reduced propensity to shed glass particulates. As a result, the presently claimed containers are particularly suited for storage of pharmaceutical compositions and, specifically, a pharmaceutical solution comprising a pharmaceutically active ingredient, for example, PEDIARIX® (Diphtheria and Tetanus Toxoids and Acellular Pertussis Adsorbed, Hepatitis B (Recombinant) and Inactivated Poliovirus Vaccine), HAVRIX® (Hepatitis A Vaccine), ENGERIX-B® (Hepatitis B Vaccine (Recombinant)), TWINRIX® (Hepatitis A &amp; Hepatitis B (Recombinant) Vaccine), EPERZAN® (albiglutide), MAGE-A3 Antigen-Specific Cancer Immunotherapeutic (astuprotimut-R), GSK2402968 (drisapersen), and HZ/su (herpes zoster vaccine).

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is related to U.S. Provisional Application No.61/815,648, filed Apr. 24, 2013, entitled “Delamination ResistantPharmaceutical Glass Containers Containing Active PharmaceuticalIngredients”, the entirety of which is hereby incorporated by referenceherein.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 22, 2014, isnamed 122467-02002_SL.txt and is 787 bytes in size.

FIELD OF THE INVENTION

The present specification generally relates to pharmaceutical containersand, more specifically, to chemically and mechanically durablepharmaceutical containers that are delamination resistant and formed, atleast in part, of a glass composition.

BACKGROUND

The design of a packaged pharmaceutical composition generally seeks toprovide an active pharmaceutical ingredient (API) in a suitable packagethat is convenient to use, that maintains the stability of the API overprolonged storage, and that ultimately allows for the delivery ofefficacious, stable, active, nontoxic and nondegraded API.

Most packaged formulations are complex physico-chemical systems, throughwhich the API is subject to deterioration by a variety of chemical,physical, and microbial reactions. Interactions between drugs,adjuvants, containers, and/or closures may occur, which can lead to theinactivation, decomposition and/or degradation of the API.

Historically, glass has been used as the preferred material forpackaging pharmaceuticals because of its hermeticity, optical clarityand excellent chemical durability relative to other materials.Specifically, the glass used in pharmaceutical packaging must haveadequate chemical durability so as not to affect the stability of thepharmaceutical compositions contained therein. Glasses having suitablechemical durability include those glass compositions within the ASTMstandard ‘Type 1B’ glass compositions which have a proven history ofchemical durability.

However, use of glass for such applications is limited by the mechanicalperformance of the glass. Specifically, in the pharmaceutical industry,glass breakage is a safety concern for the end user as the brokenpackage and/or the contents of the package may injure the end user.Further, non-catastrophic breakage (i.e., when the glass cracks but doesnot break) may cause the contents to lose their sterility which, inturn, may result in costly product recalls.

One approach to improving the mechanical durability of the glass packageis to thermally temper the glass package. Thermal tempering strengthensglass by inducing a surface compressive stress during rapid coolingafter forming. This technique works well for glass articles with flatgeometries (such as windows), glass articles with thicknesses >2 mm, andglass compositions with high thermal expansion. However, pharmaceuticalglass packages typically have complex geometries (vial, tubular,ampoule, etc.), thin walls (˜1-1.5 mm), and are produced from lowexpansion glasses (30-55×10⁻⁷K⁻¹) making glass pharmaceutical packagesunsuitable for strengthening by thermal tempering.

Chemical tempering also strengthens glass by the introduction of surfacecompressive stress. The stress is introduced by submerging the articlein a molten salt bath. As ions from the glass are replaced by largerions from the molten salt, a compressive stress is induced in thesurface of the glass. The advantage of chemical tempering is that it canbe used on complex geometries, thin samples, and is relativelyinsensitive to the thermal expansion characteristics of the glasssubstrate. However, glass compositions which exhibit a moderatesusceptibility to chemical tempering generally exhibit poor chemicaldurability and vice-versa.

Finally, glass compositions commonly used in pharmaceutical packages,e.g., Type 1a and Type 1b glass, further suffer from a tendency for theinterior surfaces of the pharmaceutical package to shed glassparticulates or “delaminate” following exposure to pharmaceuticalsolutions. Such delamination often destabilizes the activepharmaceutical ingredient (API) present in the solution, therebyrendering the API therapeutically ineffective or unsuitable fortherapeutic use.

Delamination has caused the recall of multiple drug products over thelast few years (see, for example, Reynolds et al., (2011) BioProcessInternational 9(11) pp. 52-57). In response to the growing delaminationproblem, the U.S. Food and Drug Administration (FDA) has issued anadvisory indicating that the presence of glass particulate in injectabledrugs can pose a risk.

The advisory states that, “[t]here is potential for drugs administeredintravenously that contain these fragments to cause embolic, thromboticand other vascular events; and subcutaneously to the development offoreign body granuloma, local injections site reactions and increasedimmunogenicity.”

Accordingly, a recognized need exists for alternative glass containersfor packaging of pharmaceutical compositions which exhibit a reducedpropensity to delaminate.

SUMMARY

In one aspect, the present invention is directed to a delaminationresistant pharmaceutical container formed, at least in part, of a glasscomposition including from about 70 mol. % to about 80 mol. % SiO₂; fromabout 3 mol. % to about 13 mol. % alkaline earth oxide; X mol. % Al₂O₃;and Y mol. % alkali oxide, wherein the alkali oxide includes Na₂O in anamount greater than about 8 mol. %, wherein the ratio of Y:X is greaterthan 1, and the glass composition is free of boron and compounds ofboron.

In one embodiment, the SiO₂ is present in an amount less than or equalto 78 mol. %.

In one embodiment, the amount of the alkaline earth oxide is greaterthan or equal to about 4 mol. % and less than or equal to about 8 mol.%. In a particular embodiment, the alkaline earth oxide includes MgO andCaO and has a ratio (CaO (mol. %)/(CaO (mol. %)+MgO (mol. %))) that isless than or equal to 0.5. In a particular embodiment, the alkalineearth oxide includes from about 0.1 mol. % to less than or equal toabout 1.0 mol. % CaO. In a particular embodiment, the alkaline earthoxide includes from about 3 mol. % to about 7 mol. % MgO.

In another embodiment, the alkali oxide includes greater than or equalto about 9 mol. % Na₂O and less than or equal to about 15 mol. % Na₂O.In another embodiment, the alkali oxide further includes K₂O in anamount less than or equal to about 3 mol. %. In a particular embodiment,the alkali oxide includes K₂O in an amount greater than or equal toabout 0.01 mol. % and less than or equal to about 1.0 mol. %.

In one embodiment, X is greater than or equal to about 2 mol. % and lessthan or equal to about 10 mol. %. In a particular embodiment, the ratioof Y:X is less than or equal to 2. In a particular embodiment, the ratioof Y:X is greater than or equal to 1.3 and less than or equal to 2.0.

In another embodiment, the glass composition is free of phosphorous andcompounds of phosphorous.

In one embodiment, the glass composition has a type HGB1 hydrolyticresistance according to ISO 719. Alternatively or in addition, the glasscomposition has a type HGA1 hydrolytic resistance according to ISO 720after ion exchange strengthening. Alternatively or in addition, theglass composition has a type HGA1 hydrolytic resistance according to ISO720 before and after ion exchange strengthening. Alternatively or inaddition, the glass composition has at least a class S3 acid resistanceaccording to DIN 12116. Alternatively or in addition, the glasscomposition has at least a class A2 base resistance according to ISO695.

In one embodiment, the glass composition is ion exchange strengthened.

In another embodiment, the composition further includes a compressivestress layer with a depth of layer greater than or equal to 10 μm and asurface compressive stress greater than or equal to 250 MPa.

In another aspect, the present invention provides a delaminationresistant pharmaceutical container formed, at least in part, of a glasscomposition including from about 72 mol. % to about 78 mol. % SiO₂; fromabout 4 mol. % to about 8 mol. % alkaline earth oxide; X mol. % Al₂O₃,wherein X is greater than or equal to about 4 mol. % and less than orequal to about 8 mol. %; and Y mol. % alkali oxide, wherein the alkalioxide includes Na₂O in an amount greater than or equal to about 9 mol. %and less than or equal to about 15 mol. %, wherein the ratio of Y:X isgreater than 1, and the glass composition is free of boron and compoundsof boron.

In a particular embodiment, the ratio of Y:X is less than or equal toabout 2. In a particular embodiment, the ratio of Y:X is greater than orequal to about 1.3 and less than or equal to about 2.0.

In one embodiment, the alkaline earth oxide includes MgO and CaO and hasa ratio (CaO (mol. %)/(CaO (mol. %)+MgO (mol. %))) less than or equal to0.5.

In another embodiment, the alkali oxide includes K₂O in an amountgreater than or equal to about 0.01 mol. % and less than or equal toabout 1.0 mol. %.

In another aspect, the present invention provides a delaminationresistant pharmaceutical container formed, at least in part, of a glasscomposition including from about 68 mol. % to about 80 mol. % SiO₂; fromabout 3 mol. % to about 13 mol. % alkaline earth oxide; X mol. % Al₂O₃;Y mol. % alkali oxide, wherein the alkali oxide includes Na₂O in anamount greater than about 8 mol. %; and B₂O₃, wherein the ratio (B₂O₃(mol. %)/(Y mol. %−X mol. %) is greater than 0 and less than 0.3, andthe ratio of Y:X is greater than 1.

In one embodiment, the amount of SiO₂ is greater than or equal to about70 mol. %.

In one embodiment, the amount of alkaline earth oxide is greater than orequal to about 4 mol. % and less than or equal to about 8 mol. %. In aparticular embodiment, the alkaline earth oxide includes MgO and CaO andhas a ratio (CaO (mol. %)/(CaO (mol. %)+MgO (mol. %))) less than orequal to 0.5. In a particular embodiment, the alkaline earth oxideincludes CaO in an amount greater than or equal to about 0.1 mol. % andless than or equal to about 1.0 mol. %. In a particular embodiment, thealkaline earth oxide includes from about 3 mol. % to about 7 mol. % MgO.

In one embodiment, the alkali oxide is greater than or equal to about 9mol. % Na₂O and less than or equal to about 15 mol. % Na₂O. In aparticular embodiment, the alkali oxide further includes K₂O in aconcentration less than or equal to about 3 mol. %. In anotherembodiment, the alkali oxide further includes K₂O in a concentrationgreater than or equal to about 0.01 mol. % and less than or equal toabout 1.0 mol. %.

In another embodiment, the pharmaceutical container has a ratio (B₂O₃(mol. %)/(Y mol. %−X mol. %) less than 0.2. In a particular embodiment,the amount of B₂O₃ is less than or equal to about 4.0 mol. %. In anotherembodiment, the amount of B₂O₃ is greater than or equal to about 0.01mol. %.

In one embodiment, X is greater than or equal to about 2 mol. % and lessthan or equal to about 10 mol. %. In a particular embodiment, the ratioof Y:X is less than or equal to 2. In another embodiment, the ratio ofY:X is greater than 1.3.

In one embodiment, the glass composition is free of phosphorous andcompounds of phosphorous.

In one embodiment, the glass composition has a type HGB1 hydrolyticresistance according to ISO 719. Alternatively or in addition, the glasscomposition has a type HGA1 hydrolytic resistance according to ISO 720after ion exchange strengthening. Alternatively or in addition, theglass composition has a type HGA1 hydrolytic resistance according to ISO720 before and after ion exchange strengthening. Alternatively or inaddition, the glass composition has at least a class S3 acid resistanceaccording to DIN 12116. Alternatively or in addition, the glasscomposition has at least a class A2 base resistance according to ISO695.

In one embodiment, the glass composition is ion exchange strengthened.

In another embodiment, the composition further includes a compressivestress layer with a depth of layer greater than or equal to 10 μm and asurface compressive stress greater than or equal to 250 MPa.

In one embodiment of any of the foregoing aspects of the invention, thepharmaceutical container further includes a pharmaceutical compositionhaving an active pharmaceutical ingredient. In a particular embodiment,the pharmaceutical composition includes a citrate or phosphate buffer,for example, sodium citrate, SSC, monosodium phosphate or disodiumphosphate. Alternatively or in addition, the pharmaceutical compositionhas a pH between about 7 and about 11, between about 7 and about 10,between about 7 and about 9, or between about 7 and about 8.

In one embodiment of any of the foregoing aspects of the invention, theactive pharmaceutical ingredients are diphtheria toxoid, tetanus toxoid,inactivated pertussis toxin, hepatitis B virus surface antigen,inactivated Type I poliovirus (Mahoney), Type 2 poliovirus (MEF-1), Type3 poliovirus (Saukett), filamentous hemagglutinin and pertactin (69 kDouter membrane protein), or analogs thereof. In one embodiment, thepharmaceutical composition is PEDIARIX® (Diphtheria and Tetanus Toxoidsand Acellular Pertussis Adsorbed, Hepatitis B (Recombinant) andInactivated Poliovirus Vaccine).

In one embodiment of any of the foregoing aspects of the invention, theactive pharmaceutical ingredient is inactivated hepatitis A virus, or ananalog thereof. In a particular embodiment, the pharmaceuticalcomposition is HAVRIX® (Hepatitis A Vaccine).

In one embodiment of any of the foregoing aspects of the invention, theactive pharmaceutical ingredient is noninfectious hepatitis B virussurface antigen (HBsAg), or an analog thereof. In a particularembodiment, the pharmaceutical composition is ENGERIX-B® (Hepatitis BVaccine (Recombinant)).

In one embodiment of any of the foregoing aspects of the invention, theactive pharmaceutical ingredients are inactivated hepatitis A virus andnoninfectious hepatitis B virus surface antigen (HBsAg), or analogsthereof. In a particular embodiment, the pharmaceutical composition isTWINRIX® (Hepatitis A & Hepatitis B (Recombinant) Vaccine).

In one embodiment of any of the foregoing aspects of the invention, theactive pharmaceutical ingredient is a glucagon-like peptide-1(GLP-1)receptor agonist. In a particular embodiment, the pharmaceuticalcomposition is EPERZAN® (albiglutide).

In one embodiment of any of the foregoing aspects of the invention, theactive pharmaceutical ingredient is a melanoma associated antigen 3(MAGE-A3) epitope fusion protein. In a particular embodiment, thepharmaceutical composition is MAGE-A3 Antigen-Specific CancerImmunotherapeutic (astuprotimut-R).

In one embodiment of any of the foregoing aspects of the invention, theactive pharmaceutical ingredient is a dystrophin antisenseoligonucleotide of SEQ ID NO. 1. In a particular embodiment, thepharmaceutical composition is GSK2402968 (drisapersen).

In one embodiment of any of the foregoing aspects of the invention, theactive pharmaceutical ingredient is a recombinant varicella zoster virusglycoprotein E. In a particular embodiment, the pharmaceuticalcomposition is HZ/su (herpes zoster vaccine).

In a particular aspect, the present invention provides a delaminationresistant pharmaceutical container formed, at least in part, of a glasscomposition including about 76.8 mol. % SiO₂; about 6.0 mol. % Al₂O₃;about 11.6 mol. % Na₂O; about 0.1 mol. % K₂O; about 4.8 mol. % MgO; andabout 0.5 mol. % CaO, wherein the glass composition is free of boron andcompounds of boron; and wherein the pharmaceutical container furthercomprises a pharmaceutical composition selected from the groupconsisting of PEDIARIX® (Diphtheria and Tetanus Toxoids and AcellularPertussis Adsorbed, Hepatitis B (Recombinant) and Inactivated PoliovirusVaccine), HAVRIX® (Hepatitis A Vaccine), ENGERIX-B® (Hepatitis B Vaccine(Recombinant)), TWINRIX® (Hepatitis A & Hepatitis B (Recombinant)Vaccine), EPERZAN® (albiglutide), MAGE-A3 Antigen-Specific CancerImmunotherapeutic (astuprotimut-R), GSK2402968 (drisapersen), and HZ/su(herpes zoster vaccine).

In one aspect, the present invention includes a delamination resistantpharmaceutical container including a glass composition. Thepharmaceutical container includes from about 70 mol. % to about 80 mol.% SiO₂; from about 3 mol. % to about 13 mol. % alkaline earth oxide; Xmol. % Al₂O₃; and Y mol. % alkali oxide. The alkali oxide includes Na₂Oin an amount greater than about 8 mol. %, a ratio of Y:X is greater than1, and the glass composition is free of boron and compounds of boron.The delamination resistant pharmaceutical container further includes anactive pharmaceutical ingredient.

In one or more embodiments, the SiO₂ is present in an amount less thanor equal to 78 mol. %. In some embodiments, an amount of the alkalineearth oxide is greater than or equal to about 4 mol. % and less than orequal to about 8 mol. %. In one or more embodiments, the alkaline earthoxide includes MgO and CaO and a ratio (CaO (mol. %)/(CaO (mol. %)+MgO(mol. %))) is less than or equal to 0.5. In one or more embodiments, thealkaline earth oxide includes from about 0.1 mol. % to less than orequal to about 1.0 mol. % CaO. In one or more embodiments, the alkalineearth oxide includes from about 3 mol. % to about 7 mol. % MgO. In oneor more embodiments, X is greater than or equal to about 2 mol. % andless than or equal to about 10 mol. %. In embodiments, the alkali oxideincludes greater than or equal to about 9 mol. % Na₂O and less than orequal to about 15 mol. % Na₂O. In some embodiments, the ratio of Y:X isless than or equal to 2. In one or more embodiments, the ratio of Y:X isgreater than or equal to 1.3 and less than or equal to 2.0. In one ormore embodiments, the alkali oxide further includes K₂O in an amountless than or equal to about 3 mol. %. In one or more embodiments, theglass composition is free of phosphorous and compounds of phosphorous.In one or more embodiments, the alkali oxide includes K₂O in an amountgreater than or equal to about 0.01 mol. % and less than or equal toabout 1.0 mol. %.

In another aspect, the invention includes a delamination resistantpharmaceutical container including a pharmaceutical composition. Thepharmaceutical container includes an active pharmaceutical ingredient,such that the pharmaceutical container includes a glass compositionincluding SiO₂ in a concentration greater than about 70 mol. %; alkalineearth oxide including MgO and CaO, wherein CaO is present in an amountgreater than or equal to about 0.1 mol. % and less than or equal toabout 1.0 mol. %, and a ratio (CaO (mol. %)/(CaO (mol. %)+MgO (mol. %)))is less than or equal to 0.5; and Y mol. % alkali oxide, wherein thealkali oxide includes Na₂O in an amount greater than about 8 mol. %,such that the glass composition is free of boron and compounds of boron.

In another aspect, the invention includes a delamination resistantpharmaceutical container including a pharmaceutical compositionincluding an active pharmaceutical ingredient. The pharmaceuticalcontainer includes a glass composition including from about 72 mol. % toabout 78 mol. % SiO₂; from about 4 mol. % to about 8 mol. % alkalineearth oxide, wherein the alkaline earth oxide includes MgO and CaO and aratio (CaO (mol. %)/(CaO (mol. %)+MgO (mol. %))) is less than or equalto 0.5; X mol. % Al₂O₃, such that X is greater than or equal to about 4mol. % and less than or equal to about 8 mol. %; and Y mol. % alkalioxide, such that the alkali oxide includes Na₂O in an amount greaterthan or equal to about 9 mol. % and less than or equal to about 15 mol.%, a ratio of Y:X is greater than 1, and the glass composition is freeof boron and compounds of boron.

In another aspect, the invention includes a delamination resistantpharmaceutical container including a pharmaceutical compositionincluding an active pharmaceutical ingredient. The pharmaceuticalcontainer includes a glass composition. The glass composition includesfrom about 70 mol. % to about 80 mol. % SiO₂; from about 3 mol. % toabout 13 mol. % alkaline earth oxide, such that the alkaline earth oxideincludes CaO in an amount greater than or equal to about 0.1 mol. % andless than or equal to about 1.0 mol. %, MgO, and a ratio (CaO (mol.%)/(CaO (mol. %)+MgO (mol. %))) is less than or equal to 0.5; X mol. %Al₂O₃, wherein X is greater than or equal to about 2 mol. % and lessthan or equal to about 10 mol. %; and Y mol. % alkali oxide, wherein thealkali oxide includes from about 0.01 mol. % to about 1.0 mol. % K₂O anda ratio of Y:X is greater than 1, and the glass composition is free ofboron and compounds of boron.

In one or more embodiments of any of the above aspects, thepharmaceutical composition includes PEDIARIX® (Diphtheria and TetanusToxoids and Acellular Pertussis Adsorbed, Hepatitis B (Recombinant) andInactivated Poliovirus Vaccine), HAVRIX® (Hepatitis A Vaccine),ENGERIX-B® (Hepatitis B Vaccine (Recombinant)), TWINRIX® (Hepatitis A &Hepatitis B (Recombinant) Vaccine), EPERZAN® (albiglutide), MAGE-A3Antigen-Specific Cancer Immunotherapeutic (astuprotimut-R), GSK2402968(drisapersen), or HZ/su (herpes zoster vaccine).

In one aspect, the present invention includes a pharmaceuticalcomposition. The pharmaceutical composition includes PEDIARIX®(Diphtheria and Tetanus Toxoids and Acellular Pertussis Adsorbed,Hepatitis B (Recombinant) and Inactivated Poliovirus Vaccine), HAVRIX®(Hepatitis A Vaccine), ENGERIX-B® (Hepatitis B Vaccine (Recombinant)),TWINRIX® (Hepatitis A & Hepatitis B (Recombinant) Vaccine), EPERZAN®(albiglutide), MAGE-A3 Antigen-Specific Cancer Immunotherapeutic(astuprotimut-R), GSK2402968 (drisapersen), or HZ/su (herpes zostervaccine) and a pharmaceutically acceptable excipient, such that thepharmaceutical composition is contained within a glass pharmaceuticalcontainer including an internal homogeneous layer.

In one or more embodiments, the pharmaceutical container has acompressive stress greater than or equal to 150 MPa. In one or moreembodiments, the pharmaceutical container has a compressive stressgreater than or equal to 250 MPa. In one or more embodiments, thepharmaceutical container includes a depth of layer greater than 30 μm.In one or more embodiments, the depth of layer is greater than 35 μm. Inone or more embodiments, the pharmaceutical composition demonstratesincreased stability, product integrity, or efficacy.

In one aspect, the present invention includes a pharmaceuticalcomposition. The pharmaceutical composition includes PEDIARIX®(Diphtheria and Tetanus Toxoids and Acellular Pertussis Adsorbed,Hepatitis B (Recombinant) and Inactivated Poliovirus Vaccine), HAVRIX®(Hepatitis A Vaccine), ENGERIX-B® (Hepatitis B Vaccine (Recombinant)),TWINRIX® (Hepatitis A & Hepatitis B (Recombinant) Vaccine), EPERZAN®(albiglutide), MAGE-A3 Antigen-Specific Cancer Immunotherapeutic(astuprotimut-R), GSK2402968 (drisapersen), or HZ/su (herpes zostervaccine) and a pharmaceutically acceptable excipient, such that thepharmaceutical composition is contained within a glass pharmaceuticalcontainer including an internal homogeneous layer having a compressivestress greater than or equal to 150 MPa.

In one or more embodiments, the pharmaceutical container includes adepth of layer greater than 10 μm. In one or more embodiments, thepharmaceutical container includes a depth of layer greater than 25 μm.In one or more embodiments, the pharmaceutical container includes adepth of layer greater than 30 μm. In one or more embodiments, thepharmaceutical container has compressive stress greater than or equal to300 MPa. In one or more embodiments, the pharmaceutical containerincludes increased stability, product integrity, or efficacy.

In another aspect, the present technology includes a pharmaceuticalcomposition. The pharmaceutical composition includes PEDIARIX®(Diphtheria and Tetanus Toxoids and Acellular Pertussis Adsorbed,Hepatitis B (Recombinant) and Inactivated Poliovirus Vaccine), HAVRIX®(Hepatitis A Vaccine), ENGERIX-B® (Hepatitis B Vaccine (Recombinant)),TWINRIX® (Hepatitis A & Hepatitis B (Recombinant) Vaccine), EPERZAN®(albiglutide), MAGE-A3 Antigen-Specific Cancer Immunotherapeutic(astuprotimut-R), GSK2402968 (drisapersen), or HZ/su (herpes zostervaccine) and a pharmaceutically acceptable excipient, such that thepharmaceutical composition is contained within a glass pharmaceuticalcontainer having a compressive stress greater than or equal to 150 MPaand a depth of layer greater than 10 μm, and such that thepharmaceutical composition demonstrates increased stability, productintegrity, or efficacy.

In another aspect, the present technology includes a pharmaceuticalcomposition. The pharmaceutical composition includes PEDIARIX®(Diphtheria and Tetanus Toxoids and Acellular Pertussis Adsorbed,Hepatitis B (Recombinant) and Inactivated Poliovirus Vaccine), HAVRIX®(Hepatitis A Vaccine), ENGERIX-B® (Hepatitis B Vaccine (Recombinant)),TWINRIX® (Hepatitis A & Hepatitis B (Recombinant) Vaccine), EPERZAN®(albiglutide), MAGE-A3 Antigen-Specific Cancer Immunotherapeutic(astuprotimut-R), GSK2402968 (drisapersen), or HZ/su (herpes zostervaccine) and a pharmaceutically acceptable excipient, such that thepharmaceutical composition is contained within a glass pharmaceuticalcontainer including a substantially homogeneous inner layer, and suchthat the pharmaceutical composition demonstrates increased stability,product integrity, or efficacy.

In another aspect, the present technology includes a pharmaceuticalcomposition. The pharmaceutical composition includes PEDIARIX®(Diphtheria and Tetanus Toxoids and Acellular Pertussis Adsorbed,Hepatitis B (Recombinant) and Inactivated Poliovirus Vaccine), HAVRIX®(Hepatitis A Vaccine), ENGERIX-B® (Hepatitis B Vaccine (Recombinant)),TWINRIX® (Hepatitis A & Hepatitis B (Recombinant) Vaccine), EPERZAN®(albiglutide), MAGE-A3 Antigen-Specific Cancer Immunotherapeutic(astuprotimut-R), GSK2402968 (drisapersen), or HZ/su (herpes zostervaccine) and a pharmaceutically acceptable excipient, such that thepharmaceutical composition is contained within a glass pharmaceuticalcontainer having a delamination factor of less than 3, wherein thepharmaceutical composition demonstrates increased stability, productintegrity, or efficacy.

In another aspect, the present technology includes a pharmaceuticalcomposition. The pharmaceutical composition includes PEDIARIX®(Diphtheria and Tetanus Toxoids and Acellular Pertussis Adsorbed,Hepatitis B (Recombinant) and Inactivated Poliovirus Vaccine), HAVRIX®(Hepatitis A Vaccine), ENGERIX-B® (Hepatitis B Vaccine (Recombinant)),TWINRIX® (Hepatitis A & Hepatitis B (Recombinant) Vaccine), EPERZAN®(albiglutide), MAGE-A3 Antigen-Specific Cancer Immunotherapeutic(astuprotimut-R), GSK2402968 (drisapersen), or HZ/su (herpes zostervaccine) and a pharmaceutically acceptable excipient, such that thepharmaceutical composition is contained within a glass pharmaceuticalcontainer which is substantially free of boron, and such that thepharmaceutical composition demonstrates increased stability, productintegrity, or efficacy.

In one or more embodiments, the glass pharmaceutical container has acompressive stress greater than or equal to 150 MPa and a depth of layergreater than 25 μm. In one or more embodiments, the glass pharmaceuticalcontainer has a compressive stress greater than or equal to 300 MPa anda depth of layer greater than 35 μm. In one or more embodiments, theglass pharmaceutical container includes a substantially homogeneousinner layer. In one or more embodiments, the glass pharmaceuticalcontainer has a compressive stress greater than or equal to 150 MPa anda depth of layer greater than 25 μm.

In another aspect, the present technology includes a pharmaceuticalcomposition. The pharmaceutical composition includes PEDIARIX®(Diphtheria and Tetanus Toxoids and Acellular Pertussis Adsorbed,Hepatitis B (Recombinant) and Inactivated Poliovirus Vaccine), HAVRIX®(Hepatitis A Vaccine), ENGERIX-B® (Hepatitis B Vaccine (Recombinant)),TWINRIX® (Hepatitis A & Hepatitis B (Recombinant) Vaccine), EPERZAN®(albiglutide), MAGE-A3 Antigen-Specific Cancer Immunotherapeutic(astuprotimut-R), GSK2402968 (drisapersen), or HZ/su (herpes zostervaccine) and a pharmaceutically acceptable excipient, such that thepharmaceutical composition is contained within a glass pharmaceuticalcontainer including a delamination factor of less than 3, and such thatthe pharmaceutical composition includes increased stability, productintegrity, or efficacy.

In one or more embodiments of any of the above aspects, the containerhas a compressive stress greater than or equal to 300 MPa. In one ormore embodiments, the container has a depth of layer greater than 25 μm.In one or more embodiments, the container has a depth of layer greaterthan 30 μm. In one or more embodiments, the container has a depth oflayer of at least 35 μm. In one or more embodiments, the container has acompressive stress greater than or equal to 300 MPa. In one or moreembodiments, the container has a compressive stress greater than orequal to 350 MPa.

Additional features and advantages will be set forth in the detaileddescription which follows, and in part will be readily apparent to thoseskilled in the art from that description or recognized by practicing theembodiments described herein, including the detailed description whichfollows, the claims, as well as the appended drawings.

It is to be understood that both the foregoing general description andthe following detailed description describe various embodiments and areintended to provide an overview or framework for understanding thenature and character of the claimed subject matter. The accompanyingdrawings are included to provide a further understanding of the variousembodiments, and are incorporated into and constitute a part of thisspecification. The drawings illustrate the various embodiments describedherein, and together with the description serve to explain theprinciples and operations of the claimed subject matter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 graphically depicts the relationship between the ratio of alkalioxides to alumina (x-axis) and the strain point, annealing point, andsoftening point (y-axes) of inventive and comparative glasscompositions;

FIG. 2 graphically depicts the relationship between the ratio of alkalioxides to alumina (x-axis) and the maximum compressive stress and stresschange (y-axes) of inventive and comparative glass compositions;

FIG. 3 graphically depicts the relationship between the ratio of alkalioxides to alumina (x-axis) and hydrolytic resistance as determined fromthe ISO 720 standard (y-axis) of inventive and comparative glasscompositions;

FIG. 4 graphically depicts diffusivity D (y-axis) as a function of theratio (CaO/(CaO+MgO)) (x-axis) for inventive and comparative glasscompositions;

FIG. 5 graphically depicts the maximum compressive stress (y-axis) as afunction of the ratio (CaO/(CaO+MgO)) (x-axis) for inventive andcomparative glass compositions;

FIG. 6 graphically depicts diffusivity D (y-axis) as a function of theratio (B₂O₃/(R₂O−Al₂O₃)) (x-axis) for inventive and comparative glasscompositions; and

FIG. 7 graphically depicts the hydrolytic resistance as determined fromthe ISO 720 standard (y-axis) as a function of the ratio(B₂O₃/(R₂O−Al₂O₃)) (x-axis) for inventive and comparative glasscompositions.

DETAILED DESCRIPTION

The present invention is based, at least in part, on the identificationof a pharmaceutical container formed, at least in part, of a glasscomposition which exhibits a reduced propensity to delaminate, i.e., areduced propensity to shed glass particulates. As a result, thepresently claimed containers are particularly suited for storage,maintenance and/or delivery of therapeutically efficaciouspharmaceutical compositions and, in particular pharmaceutical solutionscomprising active pharmaceutical ingredients, for example, PEDIARIX®(Diphtheria and Tetanus Toxoids and Acellular Pertussis Adsorbed,Hepatitis B (Recombinant) and Inactivated Poliovirus Vaccine), HAVRIX®(Hepatitis A Vaccine), ENGERIX-B® (Hepatitis B Vaccine (Recombinant)),TWINRIX® (Hepatitis A & Hepatitis B (Recombinant) Vaccine), EPERZAN®(albiglutide), MAGE-A3 Antigen-Specific Cancer Immunotherapeutic(astuprotimut-R), GSK2402968 (drisapersen), and HZ/su (herpes zostervaccine).

Conventional glass containers or glass packages for containingpharmaceutical compositions are generally formed from glass compositionswhich are known to exhibit chemical durability and low thermalexpansion, such as alkali borosilicate glasses. While alkaliborosilicate glasses exhibit good chemical durability, containermanufacturers have sporadically observed silica-rich glass flakesdispersed in the solution contained in the glass containers as a resultof delamination, particularly when the solution has been stored indirect contact with the glass surface for long time periods (months toyears).

Delamination refers to a phenomenon in which glass particles arereleased from the surface of the glass following a series of leaching,corrosion, and/or weathering reactions. In general, the glass particlesare silica-rich flakes of glass which originate from the interiorsurface of the package as a result of the leaching of modifier ions intoa solution contained within the package. These flakes may generally befrom about 1 nm to 2 μm thick with a width greater than about 50 μm.

It has heretofore been hypothesized that delamination is due to thephase separation which occurs in alkali borosilicate glasses when theglass is exposed to the elevated temperatures used for reforming theglass into a container shape.

However, it is now believed that the delamination of the silica-richglass flakes from the interior surfaces of the glass containers is dueto the compositional characteristics of the glass container in itsas-formed condition. Specifically, the high silica content of alkaliborosilicate glasses increases the melting temperature of the glass.However, the alkali and borate components in the glass composition meltand/or vaporize at much lower temperatures. In particular, the boratespecies in the glass are highly volatile and evaporate from the surfaceof the glass at the high temperatures necessary to melt and form theglass.

Specifically, glass stock is reformed into glass containers at hightemperatures and in direct flames. The high temperatures cause thevolatile borate species to evaporate from portions of the surface of theglass. When this evaporation occurs within the interior volume of theglass container, the volatilized borate species are re-deposited inother areas of the glass causing compositional heterogeneities in theglass container, particularly with respect to the bulk of the glasscontainer. For example, as one end of a glass tube is closed to form thebottom or floor of the container, borate species may evaporate from thebottom portion of the tube and be re-deposited elsewhere in the tube. Asa result, the areas of the container exposed to higher temperatures havesilica-rich surfaces. Other areas of the container which are amenable toboron deposition may have a silica-rich surface with a boron-rich layerbelow the surface. Areas amenable to boron deposition are at atemperature greater than the anneal point of the glass composition butless than the hottest temperature the glass is subjected to duringreformation when the boron is incorporated into the surface of theglass. Solutions contained in the container may leach the boron from theboron-rich layer. As the boron-rich layer is leached from the glass, thesilica-rich surface begins to spall, shedding silica-rich flakes intothe solution.

DEFINITIONS

The term “softening point,” as used herein, refers to the temperature atwhich the viscosity of the glass composition is 1×10^(7.6) poise.

The term “annealing point,” as used herein, refers to the temperature atwhich the viscosity of the glass composition is 1×10¹³ poise.

The terms “strain point” and “T_(strain)” as used herein, refers to thetemperature at which the viscosity of the glass composition is 3×10¹⁴poise.

The term “CTE,” as used herein, refers to the coefficient of thermalexpansion of the glass composition over a temperature range from aboutroom temperature (RT) to about 300° C.

In the embodiments of the glass compositions described herein, theconcentrations of constituent components (e.g., SiO₂, Al₂O₃, and thelike) are specified in mole percent (mol. %) on an oxide basis, unlessotherwise specified.

The terms “free” and “substantially free,” when used to describe theconcentration and/or absence of a particular constituent component in aglass composition, means that the constituent component is notintentionally added to the glass composition. However, the glasscomposition may contain traces of the constituent component as acontaminant or tramp in amounts of less than 0.01 mol. %.

The term “chemical durability,” as used herein, refers to the ability ofthe glass composition to resist degradation upon exposure to specifiedchemical conditions. Specifically, the chemical durability of the glasscompositions described herein was assessed according to threeestablished material testing standards: DIN 12116 dated March 2001 andentitled “Testing of glass—Resistance to attack by a boiling aqueoussolution of hydrochloric acid—Method of test and classification”; ISO695:1991 entitled “Glass—Resistance to attack by a boiling aqueoussolution of mixed alkali—Method of test and classification”; and ISO720:1985 entitled “Glass—Hydrolytic resistance of glass grains at 121degrees C.—Method of test and classification.” The chemical durabilityof the glass may also be assessed according to ISO 719:1985“Glass—Hydrolytic resistance of glass grains at 98 degrees C.—Method oftest and classification,” in addition to the above referenced standards.The ISO 719 standard is a less rigorous version of the ISO 720 standardand, as such, it is believed that a glass which meets a specifiedclassification of the ISO 720 standard will also meet the correspondingclassification of the ISO 719 standard. The classifications associatedwith each standard are described in further detail herein.

Glass Compositions

Reference will now be made in detail to various embodiments ofpharmaceutical containers formed, at least in part, of glasscompositions which exhibit improved chemical and mechanical durabilityand, in particular, improved resistance to delamination. The glasscompositions may also be chemically strengthened thereby impartingincreased mechanical durability to the glass. The glass compositionsdescribed herein generally comprise silica (SiO₂), alumina (Al₂O₃),alkaline earth oxides (such as MgO and/or CaO), and alkali oxides (suchas Na₂O and/or K₂O) in amounts which impart chemical durability to theglass composition. Moreover, the alkali oxides present in the glasscompositions facilitate chemically strengthening the glass compositionsby ion exchange. Various embodiments of the glass compositions will bedescribed herein and further illustrated with reference to specificexamples.

The glass compositions described herein are alkali aluminosilicate glasscompositions which generally include a combination of SiO₂, Al₂O₃, atleast one alkaline earth oxide, and one or more alkali oxides, such asNa₂O and/or K₂O. In some embodiments, the glass compositions may be freefrom boron and compounds containing boron. The combination of thesecomponents enables a glass composition which is resistant to chemicaldegradation and is also suitable for chemical strengthening by ionexchange. In some embodiments the glass compositions may furthercomprise minor amounts of one or more additional oxides such as, forexample, SnO₂, ZrO₂, ZnO, TiO₂, As₂O₃ or the like. These components maybe added as fining agents and/or to further enhance the chemicaldurability of the glass composition.

In the embodiments of the glass compositions described herein SiO₂ isthe largest constituent of the composition and, as such, is the primaryconstituent of the resulting glass network. SiO₂ enhances the chemicaldurability of the glass and, in particular, the resistance of the glasscomposition to decomposition in acid and the resistance of the glasscomposition to decomposition in water. Accordingly, a high SiO₂concentration is generally desired. However, if the content of SiO₂ istoo high, the formability of the glass may be diminished as higherconcentrations of SiO₂ increase the difficulty of melting the glasswhich, in turn, adversely impacts the formability of the glass. In theembodiments described herein, the glass composition generally comprisesSiO₂ in an amount greater than or equal to 67 mol. % and less than orequal to about 80 mol. % or even less than or equal to 78 mol. %. Insome embodiments, the amount of SiO₂ in the glass composition may begreater than about 68 mol. %, greater than about 69 mol. % or evengreater than about 70 mol. %. In some other embodiments, the amount ofSiO₂ in the glass composition may be greater than 72 mol. %, greaterthan 73 mol. % or even greater than 74 mol. %. For example, in someembodiments, the glass composition may include from about 68 mol. % toabout 80 mol. % or even to about 78 mol. % SiO₂. In some otherembodiments the glass composition may include from about 69 mol. % toabout 80 mol. % or even to about 78 mol. % SiO₂. In some otherembodiments the glass composition may include from about 70 mol. % toabout 80 mol. % or even to about 78 mol. % SiO₂. In still otherembodiments, the glass composition comprises SiO₂ in an amount greaterthan or equal to 70 mol. % and less than or equal to 78 mol. %. In someembodiments, SiO₂ may be present in the glass composition in an amountfrom about 72 mol. % to about 78 mol. %. In some other embodiments, SiO₂may be present in the glass composition in an amount from about 73 mol.% to about 78 mol. %. In other embodiments, SiO₂ may be present in theglass composition in an amount from about 74 mol. % to about 78 mol. %.In still other embodiments, SiO₂ may be present in the glass compositionin an amount from about 70 mol. % to about 76 mol. %.

The glass compositions described herein further include Al₂O₃. Al₂O₃, inconjunction with alkali oxides present in the glass compositions such asNa₂O or the like, improves the susceptibility of the glass to ionexchange strengthening. In the embodiments described herein, Al₂O₃ ispresent in the glass compositions in X mol. % while the alkali oxidesare present in the glass composition in Y mol. %. The ratio Y:X in theglass compositions described herein is greater than 1 in order tofacilitate the aforementioned susceptibility to ion exchangestrengthening. Specifically, the diffusion coefficient or diffusivity Dof the glass composition relates to the rate at which alkali ionspenetrate into the glass surface during ion exchange. Glasses which havea ratio Y:X greater than about 0.9 or even greater than about 1 have agreater diffusivity than glasses which have a ratio Y:X less than 0.9.Glasses in which the alkali ions have a greater diffusivity can obtain agreater depth of layer for a given ion exchange time and ion exchangetemperature than glasses in which the alkali ions have a lowerdiffusivity. Moreover, as the ratio of Y:X increases, the strain point,anneal point, and softening point of the glass decrease, such that theglass is more readily formable. In addition, for a given ion exchangetime and ion exchange temperature, it has been found that compressivestresses induced in glasses which have a ratio Y:X greater than about0.9 and less than or equal to 2 are generally greater than thosegenerated in glasses in which the ratio Y:X is less than 0.9 or greaterthan 2. Accordingly, in some embodiments, the ratio of Y:X is greaterthan 0.9 or even greater than 1. In some embodiments, the ratio of Y:Xis greater than 0.9, or even greater than 1, and less than or equal toabout 2. In still other embodiments, the ratio of Y:X may be greaterthan or equal to about 1.3 and less than or equal to about 2.0 in orderto maximize the amount of compressive stress induced in the glass for aspecified ion exchange time and a specified ion exchange temperature.

However, if the amount of Al₂O₃ in the glass composition is too high,the resistance of the glass composition to acid attack is diminished.Accordingly, the glass compositions described herein generally includeAl₂O₃ in an amount greater than or equal to about 2 mol. % and less thanor equal to about 10 mol. %. In some embodiments, the amount of Al₂O₃ inthe glass composition is greater than or equal to about 4 mol. % andless than or equal to about 8 mol. %. In some other embodiments, theamount of Al₂O₃ in the glass composition is greater than or equal toabout 5 mol. % to less than or equal to about 7 mol. %. In some otherembodiments, the amount of Al₂O₃ in the glass composition is greaterthan or equal to about 6 mol. % to less than or equal to about 8 mol. %.In still other embodiments, the amount of Al₂O₃ in the glass compositionis greater than or equal to about 5 mol. % to less than or equal toabout 6 mol. %.

The glass compositions also include one or more alkali oxides such asNa₂O and/or K₂O. The alkali oxides facilitate the ion exchangeability ofthe glass composition and, as such, facilitate chemically strengtheningthe glass. The alkali oxide may include one or more of Na₂O and K₂O. Thealkali oxides are generally present in the glass composition in a totalconcentration of Y mol. %. In some embodiments described herein, Y maybe greater than about 2 mol. % and less than or equal to about 18 mol.%. In some other embodiments, Y may be greater than about 8 mol. %,greater than about 9 mol. %, greater than about 10 mol. % or evengreater than about 11 mol. %. For example, in some embodiments describedherein Y is greater than or equal to about 8 mol. % and less than orequal to about 18 mol. %. In still other embodiments, Y may be greaterthan or equal to about 9 mol. % and less than or equal to about 14 mol.%.

The ion exchangeability of the glass composition is primarily impartedto the glass composition by the amount of the alkali oxide Na₂Oinitially present in the glass composition prior to ion exchange.Accordingly, in the embodiments of the glass compositions describedherein, the alkali oxide present in the glass composition includes atleast Na₂O. Specifically, in order to achieve the desired compressivestress and depth of layer in the glass composition upon ion exchangestrengthening, the glass compositions include Na₂O in an amount fromabout 2 mol. % to about 15 mol. % based on the molecular weight of theglass composition. In some embodiments the glass composition includes atleast about 8 mol. % of Na₂O based on the molecular weight of the glasscomposition. For example, the concentration of Na₂O may be greater than9 mol. %, greater than 10 mol. % or even greater than 11 mol. %. In someembodiments, the concentration of Na₂O may be greater than or equal to 9mol. % or even greater than or equal to 10 mol. %. For example, in someembodiments the glass composition may include Na₂O in an amount greaterthan or equal to about 9 mol. % and less than or equal to about 15 mol.% or even greater than or equal to about 9 mol. % and less than or equalto 13 mol. %.

As noted above, the alkali oxide in the glass composition may furtherinclude K₂O. The amount of K₂O present in the glass composition alsorelates to the ion exchangeability of the glass composition.Specifically, as the amount of K₂O present in the glass compositionincreases, the compressive stress obtainable through ion exchangedecreases as a result of the exchange of potassium and sodium ions.Accordingly, it is desirable to limit the amount of K₂O present in theglass composition. In some embodiments, the amount of K₂O is greaterthan or equal to 0 mol. % and less than or equal to 3 mol. %. In someembodiments, the amount of K₂O is less or equal to 2 mol. % or even lessthan or equal to 1.0 mol. %. In embodiments where the glass compositionincludes K₂O, the K₂O may be present in a concentration greater than orequal to about 0.01 mol. % and less than or equal to about 3.0 mol. % oreven greater than or equal to about 0.01 mol. % and less than or equalto about 2.0 mol. %. In some embodiments, the amount of K₂O present inthe glass composition is greater than or equal to about 0.01 mol. % andless than or equal to about 1.0 mol. %. Accordingly, it should beunderstood that K₂O need not be present in the glass composition.However, when K₂O is included in the glass composition, the amount ofK₂O is generally less than about 3 mol. % based on the molecular weightof the glass composition.

The alkaline earth oxides present in the composition improve themeltability of the glass batch materials and increase the chemicaldurability of the glass composition. In the glass compositions describedherein, the total mol. % of alkaline earth oxides present in the glasscompositions is generally less than the total mol. % of alkali oxidespresent in the glass compositions in order to improve the ionexchangeability of the glass composition. In the embodiments describedherein, the glass compositions generally include from about 3 mol. % toabout 13 mol. % of alkaline earth oxide. In some of these embodiments,the amount of alkaline earth oxide in the glass composition may be fromabout 4 mol. % to about 8 mol. % or even from about 4 mol. % to about 7mol. %.

The alkaline earth oxide in the glass composition may include MgO, CaO,SrO, BaO or combinations thereof. In some embodiments, the alkalineearth oxide includes MgO, CaO or combinations thereof. For example, inthe embodiments described herein the alkaline earth oxide includes MgO.MgO is present in the glass composition in an amount which is greaterthan or equal to about 3 mol. % and less than or equal to about 8 mol. %MgO. In some embodiments, MgO may be present in the glass composition inan amount which is greater than or equal to about 3 mol. % and less thanor equal to about 7 mol. % or even greater than or equal to 4 mol. % andless than or equal to about 7 mol. % by molecular weight of the glasscomposition.

In some embodiments, the alkaline earth oxide may further include CaO.In these embodiments CaO is present in the glass composition in anamount from about 0 mol. % to less than or equal to 6 mol. % bymolecular weight of the glass composition. For example, the amount ofCaO present in the glass composition may be less than or equal to 5 mol.%, less than or equal to 4 mol. %, less than or equal to 3 mol. %, oreven less than or equal to 2 mol. %. In some of these embodiments, CaOmay be present in the glass composition in an amount greater than orequal to about 0.1 mol. % and less than or equal to about 1.0 mol. %.For example, CaO may be present in the glass composition in an amountgreater than or equal to about 0.2 mol. % and less than or equal toabout 0.7 mol. % or even in an amount greater than or equal to about 0.3mol. % and less than or equal to about 0.6 mol. %.

In the embodiments described herein, the glass compositions aregenerally rich in MgO, (i.e., the concentration of MgO in the glasscomposition is greater than the concentration of the other alkalineearth oxides in the glass composition including, without limitation,CaO). Forming the glass composition such that the glass composition isMgO-rich improves the hydrolytic resistance of the resultant glass,particularly following ion exchange strengthening. Moreover, glasscompositions which are MgO-rich generally exhibit improved ion exchangeperformance relative to glass compositions which are rich in otheralkaline earth oxides. Specifically, glasses formed from MgO-rich glasscompositions generally have a greater diffusivity than glasscompositions which are rich in other alkaline earth oxides, such as CaO.The greater diffusivity enables the formation of a deeper depth of layerin the glass. MgO-rich glass compositions also enable a highercompressive stress to be achieved in the surface of the glass comparedto glass compositions which are rich in other alkaline earth oxides suchas CaO. In addition, it is generally understood that as the ion exchangeprocess proceeds and alkali ions penetrate more deeply into the glass,the maximum compressive stress achieved at the surface of the glass maydecrease with time. However, glasses formed from glass compositionswhich are MgO-rich exhibit a lower reduction in compressive stress thanglasses formed from glass compositions that are CaO-rich or rich inother alkaline earth oxides (i.e., glasses which are MgO-poor). Thus,MgO-rich glass compositions enable glasses which have higher compressivestress at the surface and greater depths of layer than glasses which arerich in other alkaline earth oxides.

In order to fully realize the benefits of MgO in the glass compositionsdescribed herein, it has been determined that the ratio of theconcentration of CaO to the sum of the concentration of CaO and theconcentration of MgO in mol. % (i.e., (CaO/(CaO+MgO)) should beminimized. Specifically, it has been determined that (CaO/(CaO+MgO))should be less than or equal to 0.5. In some embodiments (CaO/(CaO+MgO))is less than or equal to 0.3 or even less than or equal to 0.2. In someother embodiments (CaO/(CaO+MgO)) may even be less than or equal to 0.1.

Boron oxide (B₂O₃) is a flux which may be added to glass compositions toreduce the viscosity at a given temperature (e.g., the strain, annealand softening temperatures) thereby improving the formability of theglass. However, it has been found that additions of boron significantlydecrease the diffusivity of sodium and potassium ions in the glasscomposition which, in turn, adversely impacts the ion exchangeperformance of the resultant glass. In particular, it has been foundthat additions of boron significantly increase the time required toachieve a given depth of layer relative to glass compositions which areboron free. Accordingly, in some embodiments described herein, theamount of boron added to the glass composition is minimized in order toimprove the ion exchange performance of the glass composition.

For example, it has been determined that the impact of boron on the ionexchange performance of a glass composition can be mitigated bycontrolling the ratio of the concentration of B₂O₃ to the differencebetween the total concentration of the alkali oxides (i.e., R₂O, where Ris the alkali metals) and alumina (i.e., B₂O₃ (mol. %)/(R₂O (mol.%)−Al₂O₃ (mol. %)). In particular, it has been determined that when theratio of B₂O₃/(R₂O−Al₂O₃) is greater than or equal to about 0 and lessthan about 0.3 or even less than about 0.2, the diffusivities of alkalioxides in the glass compositions are not diminished and, as such, theion exchange performance of the glass composition is maintained.Accordingly, in some embodiments, the ratio of B₂O₃/(R₂O−Al₂O₃) isgreater than 0 and less than or equal to 0.3. In some of theseembodiments, the ratio of B₂O₃/(R₂O−Al₂O₃) is greater than 0 and lessthan or equal to 0.2. In some embodiments, the ratio of B₂O₃/(R₂O−Al₂O₃)is greater than 0 and less than or equal to 0.15 or even less than orequal to 0.1. In some other embodiments, the ratio of B₂O₃/(R₂O−Al₂O₃)may be greater than 0 and less than or equal to 0.05. Maintaining theratio B₂O₃/(R₂O−Al₂O₃) to be less than or equal to 0.3 or even less thanor equal to 0.2 permits the inclusion of B₂O₃ to lower the strain point,anneal point and softening point of the glass composition without theB₂O₃ adversely impacting the ion exchange performance of the glass.

In the embodiments described herein, the concentration of B₂O₃ in theglass composition is generally less than or equal to about 4 mol. %,less than or equal to about 3 mol. %, less than or equal to about 2 mol.%, or even less than or equal to 1 mol. %. For example, in embodimentswhere B₂O₃ is present in the glass composition, the concentration ofB₂O₃ may be greater than about 0.01 mol. % and less than or equal to 4mol. %. In some of these embodiments, the concentration of B₂O₃ may begreater than about 0.01 mol. % and less than or equal to 3 mol. % Insome embodiments, the B₂O₃ may be present in an amount greater than orequal to about 0.01 mol. % and less than or equal to 2 mol. %, or evenless than or equal to 1.5 mol. %. Alternatively, the B₂O₃ may be presentin an amount greater than or equal to about 1 mol. % and less than orequal to 4 mol. %, greater than or equal to about 1 mol. % and less thanor equal to 3 mol. % or even greater than or equal to about 1 mol. % andless than or equal to 2 mol. %. In some of these embodiments, theconcentration of B₂O₃ may be greater than or equal to about 0.1 mol. %and less than or equal to 1.0 mol. %.

While in some embodiments the concentration of B₂O₃ in the glasscomposition is minimized to improve the forming properties of the glasswithout detracting from the ion exchange performance of the glass, insome other embodiments the glass compositions are free from boron andcompounds of boron such as B₂O₃. Specifically, it has been determinedthat forming the glass composition without boron or compounds of boronimproves the ion exchangeability of the glass compositions by reducingthe process time and/or temperature required to achieve a specific valueof compressive stress and/or depth of layer.

In some embodiments of the glass compositions described herein, theglass compositions are free from phosphorous and compounds containingphosphorous including, without limitation, P₂O₅. Specifically, it hasbeen determined that formulating the glass composition withoutphosphorous or compounds of phosphorous increases the chemicaldurability of the glass composition.

In addition to the SiO₂, Al₂O₃, alkali oxides and alkaline earth oxides,the glass compositions described herein may optionally further compriseone or more fining agents such as, for example, SnO₂, As₂O₃, and/or Cl⁻(from NaCl or the like). When a fining agent is present in the glasscomposition, the fining agent may be present in an amount less than orequal to about 1 mol. % or even less than or equal to about 0.4 mol. %.For example, in some embodiments the glass composition may include SnO₂as a fining agent. In these embodiments SnO₂ may be present in the glasscomposition in an amount greater than about 0 mol. % and less than orequal to about 1 mol. % or even an amount greater than or equal to about0.01 mol. % and less than or equal to about 0.30 mol. %.

Moreover, the glass compositions described herein may comprise one ormore additional metal oxides to further improve the chemical durabilityof the glass composition. For example, the glass composition may furtherinclude ZnO, TiO₂, or ZrO₂, each of which further improves theresistance of the glass composition to chemical attack. In theseembodiments, the additional metal oxide may be present in an amountwhich is greater than or equal to about 0 mol. % and less than or equalto about 2 mol. %. For example, when the additional metal oxide is ZnO,the ZnO may be present in an amount greater than or equal to 1 mol. %and less than or equal to about 2 mol. %. When the additional metaloxide is ZrO₂ or TiO₂, the ZrO₂ or TiO₂ may be present in an amount lessthan or equal to about 1 mol. %.

Based on the foregoing, it should be understood that, in a firstexemplary embodiment, a glass composition may include: SiO₂ in aconcentration greater than about 70 mol. % and Y mol. % alkali oxide.The alkali oxide may include Na₂O in an amount greater than about 8 mol.%. The glass composition may be free of boron and compounds of boron.The concentration of SiO₂ in this glass composition may be greater thanor equal to about 72 mol. %, greater than 73 mol. % or even greater than74 mol. %. The glass composition of this first exemplary embodiment maybe free from phosphorous and compounds of phosphorous. The glasscomposition may also include X mol. % Al₂O₃. When Al₂O₃ is included, theratio of Y:X may be greater than 1. The concentration of Al₂O₃ may begreater than or equal to about 2 mol. % and less than or equal to about10 mol. %.

The glass composition of this first exemplary embodiment may alsoinclude alkaline earth oxide in an amount from about 3 mol. % to about13 mol. %. The alkaline earth oxide may include MgO and CaO. The CaO maybe present in an amount greater than or equal to about 0.1 mol. % andless than or equal to about 1.0 mol. %. A ratio (CaO (mol. %)/(CaO (mol.%)+MgO (mol. %))) may be less than or equal to 0.5.

In a second exemplary embodiment, a glass composition may include:greater than about 68 mol. % SiO₂; X mol. % Al₂O₃; Y mol. % alkalioxide; and B₂O₃. The alkali oxide may include Na₂O in an amount greaterthan about 8 mol %. A ratio (B₂O₃ (mol. %)/(Y mol. %−X mol. %) may begreater than 0 and less than 0.3. The concentration of SiO₂ in thisglass composition may be greater than or equal to about 72 mol. %,greater than 73 mol. % or even greater than 74 mol. %. The concentrationof Al₂O₃ may be greater than or equal to about 2 mol. % and less than orequal to about 10 mol. %. In this second exemplary embodiment, the ratioof Y:X may be greater than 1. When the ratio of Y:X is greater than 1,an upper bound of the ratio of Y:X may be less than or equal to 2. Theglass composition of this first exemplary embodiment may be free fromphosphorous and compounds of phosphorous.

The glass composition of this second exemplary embodiment may alsoinclude alkaline earth oxide. The alkaline earth oxide may include MgOand CaO. The CaO may be present in an amount greater than or equal toabout 0.1 mol. % and less than or equal to about 1.0 mol. %. A ratio(CaO (mol. %)/(CaO (mol. %)+MgO (mol. %))) may be less than or equal to0.5.

The concentration of B₂O₃ in this second exemplary embodiment may begreater than or equal to about 0.01 mol. % and less than or equal toabout 4 mol. %.

In a third exemplary embodiment, a glass article may have a type HgB1hydrolytic resistance according to ISO 719. The glass article mayinclude greater than about 8 mol. % Na₂O and less than about 4 mol. %B₂O₃. The glass article may further comprise X mol. % Al₂O₃ and Y mol. %alkali oxide. The ratio (B₂O₃ (mol. %)/(Y mol. %−X mol. %) may begreater than 0 and less than 0.3. The glass article of this thirdexemplary embodiment may further include a compressive stress layerhaving a surface compressive stress greater than or equal to about 250MPa. The glass article may also have at least a class S3 acid resistanceaccording to DIN 12116; at least a class A2 base resistance according toISO 695; and a type HgA1 hydrolytic resistance according to ISO 720.

In a fourth exemplary embodiment, a glass pharmaceutical package mayinclude SiO₂ in an amount greater than about 70 mol. %; X mol. % Al₂O₃;and Y mol. % alkali oxide. The alkali oxide may include Na₂O in anamount greater than about 8 mol. %. A ratio of a concentration of B₂O₃(mol. %) in the glass pharmaceutical package to (Y mol. %−X mol. %) maybe less than 0.3. The glass pharmaceutical package may also have a typeHGB1 hydrolytic resistance according to ISO 719. The concentration ofSiO₂ in the glass pharmaceutical package of this fourth exemplaryembodiment may be greater than or equal to 72 mol. % and less than orequal to about 78 mol. % or even greater than 74 mol. % and less than orequal to about 78 mol. %. The concentration of Al₂O₃ in the glasspharmaceutical may be greater than or equal to about 4 mol. % and lessthan or equal to about 8 mol. %. A ratio of Y:X may be greater than 1and less than 2.

The glass pharmaceutical package of this fourth exemplary embodiment mayalso include alkaline earth oxide in an amount from about 4 mol. % toabout 8 mol. %. The alkaline earth oxide may include MgO and CaO. TheCaO may be present in an amount greater than or equal to about 0.2 mol.% and less than or equal to about 0.7 mol. %. A ratio (CaO (mol. %)/(CaO(mol. %)+MgO (mol. %))) may be less than or equal to 0.5. The glasspharmaceutical package of this fourth exemplary embodiment may have atype HGA1 hydrolytic resistance according to ISO 720.

In a fifth exemplary embodiment, a glass composition may include fromabout 70 mol. % to about 80 mol. % SiO₂; from about 3 mol. % to about 13mol. % alkaline earth oxide; X mol. % Al₂O₃; and Y mol. % alkali oxide.The alkali oxide may include Na₂O in an amount greater than about 8 mol.%. A ratio of Y:X may be greater than 1. The glass composition may befree of boron and compounds of boron.

In a sixth exemplary embodiment, a glass composition may include fromabout 68 mol. % to about 80 mol. % SiO₂; from about 3 mol. % to about 13mol. % alkaline earth oxide; X mol. % Al₂O₃; and Y mol. % alkali oxide.The alkali oxide may include Na₂O in an amount greater than about 8 mol.%. The glass composition of this sixth exemplary embodiment may alsoinclude B₂O₃. A ratio (B₂O₃ (mol. %)/(Y mol. %−X mol. %) may be greaterthan 0 and less than 0.3. A ratio of Y:X may be greater than 1.

In a seventh exemplary embodiment, a glass composition may include fromabout 70 mol. % to about 80 mol. % SiO₂; from about 3 mol. % to about 13mol. % alkaline earth oxide; X mol. % Al₂O₃; and Y mol. % alkali oxide.The amount of Al₂O₃ in the glass composition may be greater than orequal to about 2 mol. % and less than or equal to about 10 mol. %. Thealkaline earth oxide may include CaO in an amount greater than or equalto about 0.1 mol. % and less than or equal to about 1.0 mol. %. Thealkali oxide may include from about 0.01 mol. % to about 1.0 mol. % K₂O.A ratio of Y:X may be greater than 1. The glass composition may be freeof boron and compounds of boron. The glass composition may be amenableto strengthening by ion exchange.

In a seventh exemplary embodiment, a glass composition may include SiO₂in an amount greater than about 70 mol. % and less than or equal toabout 80 mol. %; X mol. % Al₂O₃; and Y mol. % alkali oxide. The alkalioxide may include Na₂O in an amount greater than about 8 mol. %. A ratioof a concentration of B₂O₃ (mol. %) in the glass pharmaceutical packageto (Y mol. %−X mol. %) may be less than 0.3. A ratio of Y:X may begreater than 1.

In an eighth exemplary embodiment, a glass composition may include fromabout 72 mol. % to about 78 mol. % SiO₂; from about 4 mol. % to about 8mol. % alkaline earth oxide; X mol. % Al₂O₃, wherein X is greater thanor equal to about 4 mol. % and less than or equal to about 8 mol. %; andY mol. % alkali oxide, wherein the alkali oxide comprises Na₂O in anamount greater than or equal to about 9 mol. % and less than or equal toabout 15 mol. %. A ratio of a concentration of B₂O₃ (mol. %) in theglass pharmaceutical package to (Y mol. %−X mol. %) is less than 0.3. Aratio of Y:X may be greater than 1.

In a ninth exemplary embodiment, a pharmaceutical package for containinga pharmaceutical composition may include from about 70 mol. % to about78 mol. % SiO₂; from about 3 mol. % to about 13 mol. % alkaline earthoxide; X mol. % Al₂O₃, wherein X is greater than or equal to 2 mol. %and less than or equal to about 10 mol. %; and Y mol. % alkali oxide,wherein the alkali oxide comprises Na₂O in an amount greater than about8 mol. %. The alkaline earth oxide may include CaO in an amount lessthan or equal to about 6.0 mol. %. A ratio of Y:X may be greater thanabout 1. The package may be free of boron and compounds of boron and mayinclude a compressive stress layer with a compressive stress greaterthan or equal to about 250 MPa and a depth of layer greater than orequal to about 10 μm.

In a tenth exemplary embodiment, a glass article may be formed from aglass composition comprising from about 70 mol. % to about 78 mol. %SiO₂; alkaline earth oxide, wherein the alkaline earth oxide comprisesMgO and CaO and a ratio (CaO (mol. %)/(CaO (mol. %)+MgO (mol. %))) isless than or equal to 0.5; X mol. % Al₂O₃, wherein X is from about 2mol. % to about 10 mol. %; and Y mol. % alkali oxide, wherein the alkalioxide comprises Na₂O in an amount greater than about 8 mol. % and aratio of Y:X is greater than 1. The glass article may be ion exchangestrengthened with a compressive stress greater than or equal to 250 MPaand a depth of layer greater than or equal to 10 μm. The glass articlemay have a type HgA1 hydrolytic resistance according to ISO 720.

As noted above, the presence of alkali oxides in the glass compositionfacilitates chemically strengthening the glass by ion exchange.Specifically, alkali ions, such as potassium ions, sodium ions and thelike, are sufficiently mobile in the glass to facilitate ion exchange.In some embodiments, the glass composition is ion exchangeable to form acompressive stress layer having a depth of layer greater than or equalto 10 μm. In some embodiments, the depth of layer may be greater than orequal to about 25 μm or even greater than or equal to about 50 μm. Insome other embodiments, the depth of the layer may be greater than orequal to 75 μm or even greater than or equal to 100 μm. In still otherembodiments, the depth of layer may be greater than or equal to 10 μmand less than or equal to about 100 μm. The associated surfacecompressive stress may be greater than or equal to about 250 MPa,greater than or equal to 300 MPa or even greater than or equal to about350 MPa after the glass composition is treated in a salt bath of 100%molten KNO₃ at a temperature of 350° C. to 500° C. for a time period ofless than about 30 hours or even about less than 20 hours.

The glass articles formed from the glass compositions described hereinmay have a hydrolytic resistance of HGB2 or even HGB1 under ISO 719and/or a hydrolytic resistance of HGA2 or even HGA1 under ISO 720 (asdescribed further herein) in addition to having improved mechanicalcharacteristics due to ion exchange strengthening. In some embodimentsdescribed herein the glass articles may have compressive stresses whichextend from the surface into the glass article to a depth of layergreater than or equal to 10 μm, greater than or equal to 15 μm, greaterthan or equal to 20 μm, greater than or equal to 25 μm, greater than orequal to 30 μm or even greater than or equal to 35 μm. In someembodiments, the depth of layer may be greater than or equal to 40 μm oreven greater than or equal to 50 μm. The surface compressive stress ofthe glass article may be greater than or equal to 150 MPa, greater thanor equal to 200 MPa, greater than or equal to 250 MPa, greater than orequal to 350 MPa, or even greater than or equal to 400 MPa.

In one embodiment, the glass pharmaceutical container has a compressivestress greater than or equal to 150 MPa and a depth of layer greaterthan or equal to 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μmor 50 μm. In a particular embodiment, the glass pharmaceutical containerhas a compressive stress greater than or equal to 150 MPa and a depth oflayer greater than or equal to 10 μm. In a particular embodiment, theglass pharmaceutical container has a compressive stress greater than orequal to 150 MPa and a depth of layer greater than or equal to 25 μm. Ina particular embodiment, the glass pharmaceutical container has acompressive stress greater than or equal to 150 MPa and a depth of layergreater than or equal to 30 μm.

In one embodiment, the glass pharmaceutical container has a compressivestress greater than or equal to 300 MPa and a depth of layer greaterthan or equal to 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μmor 50 μm. In a particular embodiment, the glass pharmaceutical containerhas a compressive stress greater than or equal to 300 MPa and a depth oflayer greater than or equal to 25 μm. In yet another embodiment, theglass pharmaceutical container has a compressive stress greater than orequal to 300 MPa and a depth of layer greater than or equal to 30 μm. Inyet another embodiment, the glass pharmaceutical container has acompressive stress greater than or equal to 300 MPa and a depth of layergreater than or equal to 35 μm.

The glass compositions described herein facilitate achieving theaforementioned depths of layer and surface compressive stresses morerapidly and/or at lower temperatures than conventional glasscompositions due to the enhanced alkali ion diffusivity of the glasscompositions as described hereinabove. For example, the depths of layer(i.e., greater than or equal to 25 μm) and the compressive stresses(i.e., greater than or equal to 250 MPa) may be achieved by ionexchanging the glass article in a molten salt bath of 100% KNO₃ (or amixed salt bath of KNO₃ and NaNO₃) for a time period of less than orequal to 5 hours or even less than or equal to 4.5 hours. In someembodiments, these depths of layer and compressive stresses may beachieved by ion exchanging the glass article in a molten salt bath of100% KNO₃ (or a mixed salt bath of KNO₃ and NaNO₃) for a time period ofless than or equal to 4 hours or even less than or equal to 3.5 hours.Moreover, these depths of layers and compressive stresses may beachieved by ion exchanging the glass articles in a molten salt bath of100% KNO3 (or a mixed salt bath of KNO₃ and NaNO₃) at a temperature lessthan or equal to 500° C. or even less than or equal to 450° C. In someembodiments, these depths of layers and compressive stresses may beachieved by ion exchanging the glass articles in a molten salt bath of100% KNO3 (or a mixed salt bath of KNO₃ and NaNO₃) at a temperature lessthan or equal to 400° C. or even less than or equal to 350° C.

These improved ion exchange characteristics can be achieved when theglass composition has a threshold diffusivity of greater than about 16μm²/hr or even greater than or equal to 20 μm²/hr at 450° C. In someembodiments, the threshold diffusivity may be greater than or equal toabout 25 μm²/hr or even 30 μm²/hr at 450° C. In some other embodiments,the threshold diffusivity may be greater than or equal to about 35μm²/hr or even 40 μm²/hr at 450° C. In still other embodiments, thethreshold diffusivity may be greater than or equal to about 45 μm²/hr oreven 50 μm²/hr at 450° C.

The glass compositions described herein may generally have a strainpoint greater than or equal to about 525° C. and less than or equal toabout 650° C. The glasses may also have an anneal point greater than orequal to about 560° C. and less than or equal to about 725° C. and asoftening point greater than or equal to about 750° C. and less than orequal to about 960° C.

In the embodiments described herein the glass compositions have a CTE ofless than about 70×10⁻⁷K⁻¹ or even less than about 60×10⁻⁷K⁻¹. Theselower CTE values improve the survivability of the glass to thermalcycling or thermal stress conditions relative to glass compositions withhigher CTEs.

Further, as noted hereinabove, the glass compositions are chemicallydurable and resistant to degradation as determined by the DIN 12116standard, the ISO 695 standard, and the ISO 720 standard.

Specifically, the DIN 12116 standard is a measure of the resistance ofthe glass to decomposition when placed in an acidic solution. In brief,the DIN 12116 standard utilizes a polished glass sample of a knownsurface area which is weighed and then positioned in contact with aproportional amount of boiling 6M hydrochloric acid for 6 hours. Thesample is then removed from the solution, dried and weighed again. Theglass mass lost during exposure to the acidic solution is a measure ofthe acid durability of the sample with smaller numbers indicative ofgreater durability. The results of the test are reported in units ofhalf-mass per surface area, specifically mg/dm². The DIN 12116 standardis broken into individual classes. Class S1 indicates weight losses ofup to 0.7 mg/dm²; Class S2 indicates weight losses from 0.7 mg/dm² up to1.5 mg/dm²; Class S3 indicates weight losses from 1.5 mg/dm² up to 15mg/dm²; and Class S4 indicates weight losses of more than 15 mg/dm².

The ISO 695 standard is a measure of the resistance of the glass todecomposition when placed in a basic solution. In brief, the ISO 695standard utilizes a polished glass sample which is weighed and thenplaced in a solution of boiling 1M NaOH+0.5M Na₂CO₃ for 3 hours. Thesample is then removed from the solution, dried and weighed again. Theglass mass lost during exposure to the basic solution is a measure ofthe base durability of the sample with smaller numbers indicative ofgreater durability. As with the DIN 12116 standard, the results of theISO 695 standard are reported in units of mass per surface area,specifically mg/dm². The ISO 695 standard is broken into individualclasses. Class A1 indicates weight losses of up to 75 mg/dm²; Class A2indicates weight losses from 75 mg/dm² up to 175 mg/dm²; and Class A3indicates weight losses of more than 175 mg/dm².

The ISO 720 standard is a measure of the resistance of the glass todegradation in purified, CO₂-free water. In brief, the ISO 720 standardprotocol utilizes crushed glass grains which are placed in contact withthe purified, CO₂-free water under autoclave conditions (121° C., 2 atm)for 30 minutes. The solution is then titrated colorimetrically withdilute HCl to neutral pH. The amount of HCl required to titrate to aneutral solution is then converted to an equivalent of Na₂O extractedfrom the glass and reported in μg Na₂O per weight of glass with smallervalues indicative of greater durability. The ISO 720 standard is brokeninto individual types. Type HGA1 is indicative of up to 62 μg extractedequivalent of Na₂O per gram of glass tested; Type HGA2 is indicative ofmore than 62 μg and up to 527 μg extracted equivalent of Na₂O per gramof glass tested; and Type HGA3 is indicative of more than 527 μg and upto 930 μg extracted equivalent of Na₂O per gram of glass tested.

The ISO 719 standard is a measure of the resistance of the glass todegradation in purified, CO₂-free water. In brief, the ISO 719 standardprotocol utilizes crushed glass grains which are placed in contact withthe purified, CO₂-free water at a temperature of 98° C. at 1 atmospherefor 30 minutes. The solution is then titrated colorimetrically withdilute HCl to neutral pH. The amount of HCl required to titrate to aneutral solution is then converted to an equivalent of Na₂O extractedfrom the glass and reported in μg Na₂O per weight of glass with smallervalues indicative of greater durability. The ISO 719 standard is brokeninto individual types. The ISO 719 standard is broken into individualtypes. Type HGB1 is indicative of up to 31 μg extracted equivalent ofNa₂O; Type HGB2 is indicative of more than 31 μg and up to 62 μgextracted equivalent of Na₂O; Type HGB3 is indicative of more than 62 μgand up to 264 μg extracted equivalent of Na₂O; Type HGB4 is indicativeof more than 264 μg and up to 620 μg extracted equivalent of Na₂O; andType HGB5 is indicative of more than 620 μg and up to 1085 μg extractedequivalent of Na₂O. The glass compositions described herein have an ISO719 hydrolytic resistance of type HGB2 or better with some embodimentshaving a type HGB1 hydrolytic resistance.

The glass compositions described herein have an acid resistance of atleast class S3 according to DIN 12116 both before and after ion exchangestrengthening with some embodiments having an acid resistance of atleast class S2 or even class S1 following ion exchange strengthening. Insome other embodiments, the glass compositions may have an acidresistance of at least class S2 both before and after ion exchangestrengthening with some embodiments having an acid resistance of classS1 following ion exchange strengthening. Further, the glass compositionsdescribed herein have a base resistance according to ISO 695 of at leastclass A2 before and after ion exchange strengthening with someembodiments having a class A1 base resistance at least after ionexchange strengthening. The glass compositions described herein alsohave an ISO 720 type HGA2 hydrolytic resistance both before and afterion exchange strengthening with some embodiments having a type HGA1hydrolytic resistance after ion exchange strengthening and some otherembodiments having a type HGA1 hydrolytic resistance both before andafter ion exchange strengthening. The glass compositions describedherein have an ISO 719 hydrolytic resistance of type HGB2 or better withsome embodiments having a type HGB1 hydrolytic resistance. It should beunderstood that, when referring to the above referenced classificationsaccording to DIN 12116, ISO 695, ISO 720 and ISO 719, a glasscomposition or glass article which has “at least” a specifiedclassification means that the performance of the glass composition is asgood as or better than the specified classification. For example, aglass article which has a DIN 12116 acid resistance of “at least classS2” may have a DIN 12116 classification of either S1 or S2.

The glass compositions described herein are formed by mixing a batch ofglass raw materials (e.g., powders of SiO₂, Al₂O₃, alkali oxides,alkaline earth oxides and the like) such that the batch of glass rawmaterials has the desired composition. Thereafter, the batch of glassraw materials is heated to form a molten glass composition which issubsequently cooled and solidified to form the glass composition. Duringsolidification (i.e., when the glass composition is plasticallydeformable) the glass composition may be shaped using standard formingtechniques to shape the glass composition into a desired final form.Alternatively, the glass article may be shaped into a stock form, suchas a sheet, tube or the like, and subsequently reheated and formed intothe desired final form.

In order to assess the long-term resistance of the glass container todelamination, an accelerated delamination test was utilized. The test isperformed on glass containers after the containers have beenion-exchange strengthened. The test consisted of washing the glasscontainer at room temperature for 1 minute and depyrogenating thecontainer at about 320° C. for 1 hour. Thereafter a solution of 20 mMglycine with a pH of 10 in water is placed in the glass container to80-90% fill, the glass container is closed, and rapidly heated to 100°C. and then heated from 100° C. to 121° C. at a ramp rate of 1 deg/minat a pressure of 2 atmospheres. The glass container and solution areheld at this temperature for 60 minutes, cooled to room temperature at arate of 0.5 deg./min and the heating cycle and hold are repeated. Theglass container is then heated to 50° C. and held for two days forelevated temperature conditioning. After heating, the glass container isdropped from a distance of at least 18″ onto a firm surface, such as alaminated tile floor, to dislodge any flakes or particles that areweakly adhered to the inner surface of the glass container.

Thereafter, the solution contained in the glass container is analyzed todetermine the number of glass particles present per liter of solution.Specifically, the solution from the glass container is directly pouredonto the center of a Millipore Isopore Membrane filter (Millipore#ATTP02500 held in an assembly with parts #AP1002500 and #M000025A0)attached to vacuum suction to draw the solution through the filterwithin 10-15 seconds. Particulate flakes are then counted bydifferential interference contrast microscopy (DIC) in the reflectionmode as described in “Differential interference contrast (DIC)microscopy and modulation contrast microscopy” from Fundamentals oflight microscopy and digital imaging. New York: Wiley-Liss, pp 153-168.The field of view is set to approximately 1.5 mm×1.5 mm and particleslarger than 50 microns are counted manually. There are 9 suchmeasurements made in the center of each filter membrane in a 3×3 patternwith no overlap between images. A minimum of 100 mL of solution istested. As such, the solution from a plurality of small containers maybe pooled to bring the total amount of solution to 100 mL. If thecontainers contain more than 10 mL of solution, the entire amount ofsolution from the container is examined for the presence of particles.For containers having a volume greater than 10 mL containers, the testis repeated for a trial of 10 containers formed from the same glasscomposition under the same processing conditions and the result of theparticle count is averaged for the 10 containers to determine an averageparticle count. Alternatively, in the case of small containers, the testis repeated for a trial of 10 sets of 10 mL of solution, each of whichis analyzed and the particle count averaged over the 10 sets todetermine an average particle count. Averaging the particle count overmultiple containers accounts for potential variations in thedelamination behavior of individual containers.

It should be understood that the aforementioned test is used to identifyparticles which are shed from the interior wall(s) of the glasscontainer due to delamination and not tramp particles present in thecontainer from forming processes or particles which precipitate from thesolution enclosed in the glass container as a result of reactionsbetween the solution and the glass. Specifically, delamination particlesmay be differentiated from tramp glass particles due based on the aspectratio of the particle (i.e., the ratio of the width of the particle tothe thickness of the particle). Delamination produces particulate flakesor lamellae which are irregularly shaped and are typically >50 μm indiameter but often >200 μm. The thickness of the flakes is usuallygreater than about 100 nm and may be as large as about 1 μm. Thus, theminimum aspect ratio of the flakes is typically >50. The aspect ratiomay be greater than 100 and sometimes greater than 1000. Particlesresulting from delamination processes generally have an aspect ratiowhich is generally greater than about 50. In contrast, tramp glassparticles will generally have a low aspect ratio which is less thanabout 3. Accordingly, particles resulting from delamination may bedifferentiated from tramp particles based on aspect ratio duringobservation with the microscope. Validation results can be accomplishedby evaluating the heel region of the tested containers. Uponobservation, evidence of skin corrosion/pitting/flake removal, asdescribed in “Nondestructive Detection of Glass Vial Inner SurfaceMorphology with Differential Interference Contrast Microscopy” fromJournal of Pharmaceutical Sciences 101(4), 2012, pages 1378-1384, isnoted.

In the embodiments described herein, glass containers which average lessthan 3 glass particles with a minimum width of 50 μm and an aspect ratioof greater than 50 per trial following accelerated delamination testingare considered to have a delamination factor of 3. In the embodimentsdescribed herein, glass containers which average less than 2 glassparticles with a minimum width of 50 μm and an aspect ratio of greaterthan 50 per trial following accelerated delamination testing areconsidered to have a delamination factor of 2. In the embodimentsdescribed herein, glass containers which average less than 1 glassparticle with a minimum width of 50 μm and an aspect ratio of greaterthan 50 per trial following accelerated delamination testing areconsidered to have a delamination factor of 1. In the embodimentsdescribed herein, glass containers which have 0 glass particles with aminimum width of 50 μm and an aspect ratio of greater than 50 per trialfollowing accelerated delamination testing are considered to have adelamination factor of 0. Accordingly, it should be understood that thelower the delamination factor, the better the resistance of the glasscontainer to delamination. In the embodiments described herein, theglass containers have a delamination factor of 3 or lower (i.e., adelamination factor of 3, 2, 1 or 0).

Pharmaceutical Containers

In view of the chemical durability of the glass composition of thepresent invention, the glass compositions described herein areparticularly well suited for use in designing pharmaceutical containersfor storing, maintaining and/or delivering pharmaceutical compositions,such as liquids, solutions, powders, e.g., lyophilized powders, solidsand the like. As used herein, the term “pharmaceutical container” refersto a composition designed to store, maintain and/or deliver apharmaceutical composition. The pharmaceutical containers, as describedherein, are formed, at least in part, of the delamination resistantglass compositions described above. Pharmaceutical containers of thepresent invention include, but are not limited to, Vacutainers™cartridges, syringes, ampoules, bottles, flasks, phials, tubes, beakers,vials, injection pens or the like. In a particular embodiment, thepharmaceutical container is a vial. In a particular embodiment, thepharmaceutical container is an ampoule. In a particular embodiment, thepharmaceutical container is an injection pen. In a particularembodiment, the pharmaceutical container is a tube. In a particularembodiment, the pharmaceutical container is a bottle. In a particularembodiment, the pharmaceutical container is a syringe.

Moreover, the ability to chemically strengthen the glass compositionsthrough ion exchange can be utilized to improve the mechanicaldurability of pharmaceutical containers formed from the glasscomposition. Accordingly, it should be understood that, in at least oneembodiment, the glass compositions are incorporated in a pharmaceuticalcontainer in order to improve the chemical durability and/or themechanical durability of the pharmaceutical container.

Pharmaceutical Compositions

In various embodiments, the pharmaceutical container further includes apharmaceutical composition comprising an active pharmaceuticalingredient (API). As used herein, the term “pharmaceutical composition”refers to a composition comprising an active pharmaceutical ingredientto be delivered to a subject, for example, for therapeutic,prophylactic, diagnostic, preventative or prognostic effect. In certainembodiments, the pharmaceutical composition comprises the activepharmaceutical ingredient and a pharmaceutically acceptable carrier. Asused herein, “pharmaceutically acceptable carrier” includes any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Examples of pharmaceutically acceptablecarriers include one or more of water, saline, phosphate bufferedsaline, dextrose, glycerol, ethanol and the like, as well ascombinations thereof. In many cases, it may be preferable to includeisotonic agents, for example, sugars, polyalcohols such as mannitol,sorbitol, or sodium chloride in the composition. Pharmaceuticallyacceptable carriers may further comprise minor amounts of auxiliarysubstances such as wetting or emulsifying agents, preservatives orbuffers, which enhance the shelf life or effectiveness of the activepharmaceutical agent.

As used herein, the term “active pharmaceutical ingredient” or “API”refers a substance in a pharmaceutical composition that provides adesired effect, for example, a therapeutic, prophylactic, diagnostic,preventative or prognostic effect. In various embodiments, the activepharmaceutical ingredient can be any of a variety of substances known inthe art, for example, a small molecule, a polypeptide mimetic, abiologic, an antisense RNA, a small interfering RNA (siRNA), etc.

For example, in a particular embodiment, the active pharmaceuticalingredient may be a small molecule. As used herein, the term “smallmolecule” includes any chemical or other moiety, other than polypeptidesand nucleic acids, that can act to affect biological processes. Smallmolecules can include any number of therapeutic agents presently knownand used, or that can be synthesized from a library of such moleculesfor the purpose of screening for biological function(s). Small moleculesare distinguished from macromolecules by size. The small molecules ofthe present invention usually have a molecular weight less than about5,000 daltons (Da), preferably less than about 2,500 Da, more preferablyless than 1,000 Da, most preferably less than about 500 Da.

Small molecules include, without limitation, organic compounds,peptidomimetics and conjugates thereof. As used herein, the term“organic compound” refers to any carbon-based compound other thanmacromolecules such as nucleic acids and polypeptides. In addition tocarbon, organic compounds may contain calcium, chlorine, fluorine,copper, hydrogen, iron, potassium, nitrogen, oxygen, sulfur and otherelements. An organic compound may be in an aromatic or aliphatic form.Non-limiting examples of organic compounds include acetones, alcohols,anilines, carbohydrates, monosaccharides, oligosaccharides,polysaccharides, amino acids, nucleosides, nucleotides, lipids,retinoids, steroids, proteoglycans, ketones, aldehydes, saturated,unsaturated and polyunsaturated fats, oils and waxes, alkenes, esters,ethers, thiols, sulfides, cyclic compounds, heterocyclic compounds,imidizoles, and phenols. An organic compound as used herein alsoincludes nitrated organic compounds and halogenated (e.g., chlorinated)organic compounds.

In another embodiment, the active pharmaceutical ingredient may be apolypeptide mimetic (“peptidomimetic”). As used herein, the term“polypeptide mimetic” is a molecule that mimics the biological activityof a polypeptide, but that is not peptidic in chemical nature. While, incertain embodiments, a peptidomimetic is a molecule that contains nopeptide bonds (that is, amide bonds between amino acids), the termpeptidomimetic may include molecules that are not completely peptidic incharacter, such as pseudo-peptides, semi-peptides, and peptoids.

In other embodiments, the active pharmaceutical ingredient may be abiologic. As used herein, the term “biologic” includes products createdby biologic processes instead of by chemical synthesis. Non-limitingexamples of a “biologic” include proteins, antibodies, antibody likemolecules, vaccines, blood, blood components, and partially purifiedproducts from tissues.

The terms “peptide,” “polypeptide,” and “protein” are usedinterchangeably herein. In the present invention, these terms mean alinked sequence of amino acids, which may be natural, synthetic, or amodification or combination of natural and synthetic. The term includesantibodies, antibody mimetics, domain antibodies, lipocalins, andtargeted proteases. The term also includes vaccines containing a peptideor peptide fragment intended to raise antibodies against the peptide orpeptide fragment.

“Antibody” as used herein includes an antibody of classes IgG, IgM, IgA,IgD, or IgE, or fragments or derivatives thereof, including Fab,F(ab′)2, Fd, and single chain antibodies, diabodies, bispecificantibodies, and bifunctional antibodies. The antibody may be amonoclonal antibody, polyclonal antibody, affinity purified antibody, ormixtures thereof, which exhibits sufficient binding specificity to adesired epitope or a sequence derived therefrom. The antibody may alsobe a chimeric antibody. The antibody may be derivatized by theattachment of one or more chemical, peptide, or polypeptide moietiesknown in the art. The antibody may be conjugated with a chemical moiety.The antibody may be a human or humanized antibody.

Other antibody-like molecules are also within the scope of the presentinvention. Such antibody-like molecules include, e.g., receptor traps(such as entanercept), antibody mimetics (such as adnectins, fibronectinbased “addressable” therapeutic binding molecules from, e.g., CompoundTherapeutics, Inc.), domain antibodies (the smallest functional fragmentof a naturally occurring single-domain antibody (such as, e.g.,nanobodies; see, e.g., Cortez-Retamozo et al., Cancer Res. 2004 Apr. 15;64 (8):2853-7)).

Suitable antibody mimetics generally can be used as surrogates for theantibodies and antibody fragments described herein. Such antibodymimetics may be associated with advantageous properties (e.g., they maybe water soluble, resistant to proteolysis, and/or be nonimmunogenic).For example, peptides comprising a synthetic beta-loop structure thatmimics the second complementarity-determining region (CDR) of monoclonalantibodies have been proposed and generated. See, e.g., Saragovi et al.,Science. Aug. 16, 1991; 253 (5021):792-5. Peptide antibody mimetics alsohave been generated by use of peptide mapping to determine “active”antigen recognition residues, molecular modeling, and a moleculardynamics trajectory analysis, so as to design a peptide mimic containingantigen contact residues from multiple CDRs. See, e.g., Cassett et al.,Biochem Biophys Res Commun. Jul. 18, 2003; 307 (1):198-205. Additionaldiscussion of related principles, methods, etc., that may be applicablein the context of this invention are provided in, e.g., Fassina,Immunomethods. October 1994; 5 (2):121-9.

In various embodiments, the active pharmaceutical ingredient may haveany of a variety of activities selected from the group consisting ofanti-rheumatics, anti-neoplastic, vaccines, anti-diabetics,haematologicals, muscle relaxant, immunostimulants, anti-coagulants,bone calcium regulators, sera and gammaglobulins, anti-fibrinolytics, MStherapies, anti-anaemics, cytostatics, interferons, anti-metabolites,radiopharmaceuticals, anti-psychotics, anti-bacterials,immunosuppressants, cytotoxic antibiotics, cerebral & peripheralvasotherapeutics, nootropics, CNS drugs, dermatologicals, angiotensinantagonists, anti-spasmodics, anti-cholinergics, interferons,anti-psoriasis agents, anti-hyperlipidaemics, cardiac therapies,alkylating agents, bronchodilators, anti-coagulants,anti-inflammatories, growth hormones, and diagnostic imaging agents.

In various embodiments, the pharmaceutical composition may be selectedfrom the group consisting of PEDIARIX® (Diphtheria and Tetanus Toxoidsand Acellular Pertussis Adsorbed, Hepatitis B (Recombinant) andInactivated Poliovirus Vaccine), HAVRIX® (Hepatitis A Vaccine),ENGERIX-B® (Hepatitis B Vaccine (Recombinant)), TWINRIX® (Hepatitis A &Hepatitis B (Recombinant) Vaccine), EPERZAN® (albiglutide), MAGE-A3Antigen-Specific Cancer Immunotherapeutic (astuprotimut-R), GSK2402968(drisapersen), and HZ/su (herpes zoster vaccine).

In a particular embodiment, the pharmaceutical composition comprisesDiphtheria and Tetanus Toxoids and Acellular Pertussis Adsorbed,Hepatitis B (Recombinant) and Inactivated Poliovirus Vaccine(PEDIARIX®). In a particular embodiment, the active pharmaceuticalingredient comprises diphtheria toxoid, tetanus toxoid, inactivatedpertussis toxin, hepatitis B virus surface antigen, inactivated Type Ipoliovirus (Mahoney), Type 2 poliovirus (MEF-1), Type 3 poliovirus(Saukett), filamentous hemagglutinin and pertactin (69 kD outer membraneprotein), or analogs thereof. PEDIARIX® is a form of noninfectious,sterile vaccine comprising diphtheria toxoid, tetanus toxoid,inactivated pertussis toxin, hepatitis B virus surface antigen,inactivated Type I poliovirus (Mahoney), Type 2 poliovirus (MEF-1), Type3 poliovirus (Saukett), filamentous hemagglutinin and pertactin (69 kDouter membrane protein). Protection against disease is due to thedevelopment of neutralizing antibodies to the diphtheria toxin, thetetanus toxin, and to the poliovirus. The role of the different B.pertussis components (inactivated pertussis toxin, filamentoushemagglutinin and pertactin (69 kD outer membrane protein)) in thedevelopment of immunity to pertussis is not well understood. Antibodyconcentrations >10 mIU/mL against HBsAg confer protection againsthepatitis B virus infection. PEDIARIX® is presently indicated for activeimmunization against diphtheria, tetanus, pertussis, infection caused byall known subtypes of hepatitis B virus, and poliomyelitis.

The diphtheria toxin and tetanus toxin are produced by growingCorynebacterium diphtheria and Clostridium tetani in culture,respectively, followed by detoxification of the toxins withformaldehyde. The toxins are concentrated by ultrafiltration andpurified by precipitation, dialysis and sterile filtration. Thepertussis antigens are isolated from Bordatella pertussis cells grown inculture. The pertussis toxin and filamentous hemagglutinin are isolatedfrom the medium and the pertactin is isolated from the cells. Allantigens are purified by chromatography and precipitation. The pertussistoxin is inactivated using glutaraldehye and formaldehye and thefilamentous hemagglutinin and pertactin are treated with formaldehyde.The hepatitis B surface antigen is derived from recombinantSaccharomyces cerevisiae cells which carry the surface antigen gene ofthe hepatitis B virus. The surface antigen is purified by precipitation,ion exchange chromatography and ultrafiltration. The three strains ofpoliovirus are grown individually in VERO cells followed by purificationby ultrafiltration, diafiltration and chromatography and inactivatedusing formaldehyde. The individually purified viruses are combined intoa trivalent vaccine concentrate.

PEDIARIX® is a noninfectious, sterile vaccine for intramuscularadministration. Three doses of 0.5 ml each are administered at 2, 4 and6 months of age. Each 0.5-ml dose contains 25 Lf of diphtheria toxoid,10 Lf of tetanus toxoid, 25 mcg of inactivated pertussis toxin (PT), 25mcg of filamentous hemagglutinin (FHA), 8 mcg of pertactin (69 kDa outermembrane protein), 10 mcg of HBsAg, 40 D-antigen Units (DU) of Type 1poliovirus (Mahoney), 8 DU of Type 2 poliovirus (MEF-1), and 32 DU ofType 3 poliovirus (Saukett). Each 0.5-ml dose contains not more than0.85 mg aluminum salts as adjuvant and 4.5 mg sodium chloride. Each dosealso contains ≦100 mcg of residual formaldehyde, ≦100 mcg of polysorbate80 (Tween 80), and ≦5% yeast protein. Neomycin sulfate and polymyxin Bmay be present in the final vaccine at ≦0.05 ng and ≦0.01 ng per dose,respectively. PEDIARIX® is a suspension for injection available in 0.5ml single-dose disposable prefilled syringe.

In a particular embodiment, the pharmaceutical composition comprisesHepatitis A Vaccine. In a particular embodiment, the activepharmaceutical ingredient comprises inactivated hepatitis A virus, or ananalog thereof. Hepatitis A Vaccine (HAVRIX®) is a form of sterilesuspension of inactivated hepatitis A virus (strain HM175). The presenceof antibodies to hepatitis A virus confers protection against hepatitisA disease. HAVRIX® is indicated for active immunization against diseasecaused by hepatitis A virus infection.

The hepatitis A virus is propagated in MRC-5 human diploid cells andinactivated with formalin. Viral antigen activity is referenced to astandard using an enzyme linked immunosorbent assay (ELISA) and isexpressed in terms of ELISA Units.

HAVRIX® is a sterile vaccine for intramuscular administration. Adultsreceive a single 1 ml dose and a 1 ml booster dose between 6 to 12months later. Children and adolescents receive a single 0.5 ml dose anda 0.5 ml booster dose between 6 to 12 months later. Each 1 ml dosecontains 1440 ELISA Units of viral antigen adsorbed onto 0.5 mg ofaluminum as aluminum hydroxide. Each 0.5 ml dose contains 720 ELISAUnits of viral antigen adsorbed onto 0.25 mg of aluminum as aluminumhydroxide. The dose also contains amino acid supplement (0.3% w/v) in aphosphate-buffered saline solution and polysorbate 20 (0.05 mg/ml).Residual compounds from the manufacturing process include not more than5 mcg/ml residual MRC-5 cellular sulfate, not more than 0.1 mg/mlformalin and not more than 40 ng/ml neomycin sulfate. HAVRIX® iscurrently supplied in either a 0.5 ml or 1 ml single-dose vial and in a0.5 ml or 1 ml prefilled syringe. HAVRIX® is a homogeneous turbid whitesuspension for injection.

In a particular embodiment, the pharmaceutical composition comprisesHepatitis B Vaccine (Recombinant). In a particular embodiment, theactive pharmaceutical ingredient comprises noninfectious hepatitis Bvirus surface antigen (HBsAg), or analogs thereof. Hepatitis B Vaccine(Recombinant) (ENGERIX-B®) is a form of sterile suspension ofnoninfectious hepatitis B virus surface antigen (HBsAg). Protectionagainst hepatitis B virus infection is due to antibodyconcentrations >10 mIU/mL against HBsAg. Seroconversion is defined asantibody titers ≧1 mIU/ml. ENGERIX-B® is indicated for activeimmunization against infection caused by all known subtypes of hepatitisB virus.

The hepatitis B surface antigen is derived from recombinantSaccharomyces cerevisiae cells which carry the surface antigen gene ofthe hepatitis B virus. The surface antigen is purified by precipitation,ion exchange chromatography and ultrafiltration.

ENGERIX-B® is a sterile vaccine for intramuscular administration.Individuals 20 years of age and older receive a series of 3, 1 ml dosesgiven on a 0, 1, and 6 month schedule. Individuals from birth to 19years of age receive a series of 3, 0.5 ml doses given on a 0, 1, and 6month schedule. Each 1 ml dose contains 20 mcg of HBsAg adsorbed on 0.5mg aluminum as aluminum hydroxide. Each 0.5 ml dose contains 10 mcg ofHBsAg adsorbed on 0.25 mg aluminum as aluminum hydroxide. Excipientsinclude sodium chloride (9 mg/ml) and phosphate buffers (disodiumphosphate dihydrate, 0.98 mg/ml; sodium dihydrogen phosphate dihydrate,0.71 mg/ml). Each dose contains no more than 5% yeast protein. ENGERIX®is currently supplied in either a 0.5 ml or 1 ml single-dose vial and ina 0.5 ml or 1 ml prefilled syringe. ENGERIX® is a homogeneous turbidwhite suspension for injection.

In a particular embodiment, the pharmaceutical composition comprisesHepatitis A & Hepatitis B (Recombinant) Vaccine. In a particularembodiment, the active pharmaceutical ingredient comprises inactivatedhepatitis A virus and noninfectious hepatitis B virus surface antigen(HBsAg), or analogs thereof. Hepatitis A & Hepatitis B (Recombinant)Vaccine (TWINRIX®) is a form of bivalent vaccine containing inactivatedhepatitis A virus (strain HM175) and noninfectious hepatitis B virussurface antigen (HBsAg). The presence of antibodies to hepatitis A virusconfers protection against hepatitis A disease. Protection againsthepatitis B virus infection is due to antibody concentrations >10 mIU/mLagainst HBsAg. TWINRIX® is indicated for active immunization againstdisease caused by hepatitis A virus infection and infection by all knownsubtypes of hepatitis B virus.

The hepatitis A virus is propagated in MRC-5 human diploid cells andinactivated with formalin. Viral antigen activity is referenced to astandard using an enzyme linked immunosorbent assay (ELISA) and isexpressed in terms of ELISA Units. The hepatitis B surface antigen isderived from recombinant Saccharomyces cerevisiae cells which carry thesurface antigen gene of the hepatitis B virus. The surface antigen ispurified by precipitation, ion exchange chromatography andultrafiltration.

TWINRIX® is a sterile vaccine for intramuscular administration.Individuals receive a standard dosing regimen of 3, 1 ml doses at 0, 1,and 6 months. Under an accelerated dosing regimen, individuals receive4, 1 ml doses on days 0, 7, and 21 to 30 followed by a booster dose at12 months. Each 1 ml dose contains 720 ELISA Units of inactivatedhepatitis A virus and 20 mcg of recombinant HBsAg protein. The 1 ml doseof vaccine also contains 0.45 mg of aluminum in the form of aluminumphosphate and aluminum hydroxide as adjuvants, amino acids, sodiumchloride, phosphate buffer, polysorbate 20 and Water for Injection.Residual compounds from the manufacturing process include not more than0.1 mg of formalin, not more than 2.5 mcg of MRC-5 cellular proteins,not more than 20 ng neomycin sulfate and not more than 5% yeast protein.TWINRIX® is currently supplied as a 1 ml single-dose vial and as aprefilled syringe and is formulated as a turbid white suspension forinjection.

In particular embodiments, the pharmaceutical composition comprisesalbiglutide. In a particular embodiment, the active pharmaceuticalingredient comprises a glucagon-like peptide-1 (GLP-1) receptor agonist,or an analog thereof. Albiglutide (EPERZAN®) is a form of glucagon-likepeptide-1 (GLP-1) receptor agonist. GLP-1 is secreted by thegastrointestinal tract during eating and increases insulin secretion,decreases glycemic excursion, delays gastric emptying and reduces foodintake. Albiglutide is presently indicated for treatment of adults withtype 2 diabetes.

Albiglutide is a GLP-1 receptor agonist comprising a dipeptidylpeptidase-4 resistant GLP-1 dimer fused in series to human albuminNative GLP-1 peptide is rapidly degraded while albiglutide has a longerduration of action (e.g. half-life of 4 to 7 days) due to its resistanceto hydrolysis by dipeptidyl peptidase-4. Albiglutide is designed forweekly or biweekly subcutaneous dosing. Albiglutide may be provided at aconcentration of 50 mg/mL following resuspension of a lyophilized formcomprising 2.8% mannitol, 4.2% trehalose dihydrate, 0.01% polysorbate80, and 10 to 20 mM phosphate buffer at pH 7.2. The formulation isdiluted with water for injection as necessary for dosing.

In a particular embodiment, the pharmaceutical composition comprisesastuprotimut-R. In a particular embodiment, the active pharmaceuticalingredient comprises a fusion protein consisting of the MAGE-A3(melanoma associated antigen 3), or an analog thereof. Astuprotimut-R(MAGE-A3 Antigen-Specific Cancer Immunotherapeutic) is a form of fusionprotein consisting of the MAGE-A3 (melanoma associated antigen 3)epitope. MAGE-A3 may also be referred to as MAGE-3. A MAGE-A3 fusionprotein may induce a therapeutic effect by stimulating a cytotoxic Tlymphocyte (CTL) response against tumor cells that express the MAGE-A3antigen, resulting in tumor cell death. Astuprotimut-R is presentlyindicated for the treatment of cancer (e.g., melanoma, non-small celllung cancer).

Astuprotimut-R is a fusion protein consisting of the MAGE-A3 (melanomaassociated antigen 3) epitope fused to part of the Haemophilus influenzaprotein D antigen sequence. MAGE-A3 is a tumor specific antigenexpressed in a variety of cancers (e.g., melanoma, non-small cell lungcancer, head and neck cancer and bladder cancer). Astuprotimut-R isadministered along with an adjuvant system comprising specificcombinations of immunostimulating compounds selected to increase theanti-tumor response.

In a particular embodiment, the pharmaceutical composition comprisesdrisapersen. In a particular embodiment, the active pharmaceuticalingredient is a dystrophin antisense oligonucleotide, or an analogthereof. Drisapersen (GSK2402968) is a form of dystrophin antisenseoligonucleotide. Antisense oligonucleotides may affect thepost-transcriptional splicing process of the premRNA to restore thereading frame of the mRNA, resulting in a shortened dystrophin protein.Drisapersen is currently indicated for the treatment of boys withDuchenne Muscular Dystrophy with a dystrophin gene mutation amenable toan exon 51 skip.

Drisapersen is a dystrophin antisense oligonucleotide(5′-UCAAGGAAGAUGGCAUUUCA-3′) (SEQ ID NO:1) with full length2′-O-methyl-substituted ribose moieties and phosphorothioateinternucleotide linkages. Drisapersen is designed to cause skipping ofexon 51 of the dystrophin pre-mRNA and to restore the reading frame.Binding of the antisense oligonucleotide to either one or both splicesites or exon-internal sequences induces skipping of the exon byblocking recognition of the splice sites by the spliceosome complex.

Drisapersen may be provided in 0.5 ml glass vial in sodium phosphatebuffered saline at a concentration of 100 mg/ml. It may be formulatedfor abdominal subcutaneous administration at a dose of 0.5 to 10 mg/kgof body weight.

In a particular embodiment, the pharmaceutical composition comprises aherpes zoster vaccine. In a particular embodiment, the activepharmaceutical composition comprises a recombinant varicella zostervirus glycoprotein E, or an analog thereof. Herpes zoster vaccine(HZ/su) is a form of recombinant adjuvated vaccine comprising thevaricella zoster virus glycoprotein E. The varicella zoster virusglycoprotein E subunit is the most abundant glycoprotein in thevaricella zoster virus and induces a potent CD4+ T-cell response whenadministered with an adjuvant. Herpes zoster vaccine is presentlyindicated for prevention of herpes zoster infection and itscomplications.

Herpes virus vaccine is formulated for intramuscular injection andcomprises 50 μg of varicella zoster virus glycoprotein E in 0.2 ml mixedwith 0.5 ml of AS01B adjuvant. AS01B adjuvant is a liposome basedadjuvant system containing 50 μg 3-O-desacyl-4′-monophosphoryl lipid A(MPL) and 50 μg QS21 (a triterpene glycoside).

Degradation and Stability of Pharmaceutical Compositions

According to the present invention, delamination resistantpharmaceutical containers comprising a glass composition provide forimproved resistance to degradation of, improved stability of, improvedresistance to inactivation of, and improved maintenance of levels of apharmaceutical composition having at least one active pharmaceuticalingredient, for example, PEDIARIX® (Diphtheria and Tetanus Toxoids andAcellular Pertussis Adsorbed, Hepatitis B (Recombinant) and InactivatedPoliovirus Vaccine), HAVRIX® (Hepatitis A Vaccine), ENGERIX-B®(Hepatitis B Vaccine (Recombinant)), TWINRIX® (Hepatitis A & Hepatitis B(Recombinant) Vaccine), EPERZAN® (albiglutide), MAGE-A3 Antigen-SpecificCancer Immunotherapeutic (astuprotimut-R), GSK2402968 (drisapersen), orHZ/su (herpes zoster vaccine).

In one embodiment of the present invention, the delamination resistantpharmaceutical containers provide improved stability to pharmaceuticalcompositions contained therein, for example, PEDIARIX® (Diphtheria andTetanus Toxoids and Acellular Pertussis Adsorbed, Hepatitis B(Recombinant) and Inactivated Poliovirus Vaccine), HAVRIX® (Hepatitis AVaccine), ENGERIX-B® (Hepatitis B Vaccine (Recombinant)), TWINRIX®(Hepatitis A & Hepatitis B (Recombinant) Vaccine), EPERZAN®(albiglutide), MAGE-A3 Antigen-Specific Cancer Immunotherapeutic(astuprotimut-R), GSK2402968 (drisapersen), or HZ/su (herpes zostervaccine). As used herein, the term “stability” refers to the ability ofan active pharmaceutical ingredient to essentially retain its physical,chemical and conformational identity and integrity upon storage in thepharmaceutical containers of the invention. Stability is associated withthe ability of an active pharmaceutical ingredient to retain its potencyand efficacy over a period of time. Instability of an activepharmaceutical ingredient may be associated with, for example, chemicalor physical degradation, fragmentation, conformational change, increasedtoxicity, aggregation (e.g., to form higher order polymers),deglycosylation, modification of glycosylation, oxidation, hydrolysis,or any other structural, chemical or physical modification. Suchphysical, chemical and/or conformational changes often result in reducedactivity or inactivation of the active pharmaceutical ingredient, forexample, such that at least one biological activity of the activepharmaceutical ingredient is reduced or eliminated. Alternatively or inaddition, such physical, chemical and/or conformational changes oftenresult in the formation of structures toxic to the subject to whom thepharmaceutical composition is administered.

The pharmaceutical containers of the present invention maintainstability of the pharmaceutical compositions, in part, by minimizing oreliminating delamination of the glass composition which forms, at leastin part, the pharmaceutical container. In addition, the pharmaceuticalcontainers of the present invention maintain stability of thepharmaceutical compositions, in part, by reducing or preventing theinteraction of the active pharmaceutical ingredient with thepharmaceutical container and/or delaminated particles resultingtherefrom. By minimizing or eliminating delamination and, further, byreducing or preventing interaction, the pharmaceutical containersthereby reduce or prevent the destabilization of the activepharmaceutical ingredient as found in, for example, PEDIARIX®(Diphtheria and Tetanus Toxoids and Acellular Pertussis Adsorbed,Hepatitis B (Recombinant) and Inactivated Poliovirus Vaccine), HAVRIX®(Hepatitis A Vaccine), ENGERIX-B® (Hepatitis B Vaccine (Recombinant)),TWINRIX® (Hepatitis A & Hepatitis B (Recombinant) Vaccine), EPERZAN®(albiglutide), MAGE-A3 Antigen-Specific Cancer Immunotherapeutic(astuprotimut-R), GSK2402968 (drisapersen), or HZ/su (herpes zostervaccine).

The pharmaceutical containers of the present invention provide theadditional advantage of preventing loss of active pharmaceuticalingredients. For example, by reducing or preventing the interaction ofand, thus, the adherence of, the active pharmaceutical ingredient withthe pharmaceutical container and/or delaminated particles resultingtherefrom, the level of active pharmaceutical ingredient available foradministration to a subject is maintained, as found in, for example,PEDIARIX® (Diphtheria and Tetanus Toxoids and Acellular PertussisAdsorbed, Hepatitis B (Recombinant) and Inactivated Poliovirus Vaccine),HAVRIX® (Hepatitis A Vaccine), ENGERIX-B® (Hepatitis B Vaccine(Recombinant)), TWINRIX® (Hepatitis A & Hepatitis B (Recombinant)Vaccine), EPERZAN® (albiglutide), MAGE-A3 Antigen-Specific CancerImmunotherapeutic (astuprotimut-R), GS K2402968 (drisapersen), or HZ/su(herpes zoster vaccine).

In one embodiment of the present invention, the pharmaceuticalcomposition has a high pH. According to the present invention, it hasbeen discovered that high pHs serve to increase delamination of glasscompositions. Accordingly, the pharmaceutical containers of the presentinvention are particularly suitable for storing and maintainingpharmaceutical compositions having a high pH, for example,pharmaceutical compositions having a pH between about 7 and about 11,between about 7 and about 10, between about 7 and about 9, or betweenabout 7 and about 8.

In additional embodiments, the pharmaceutical containers of the presentinvention are particularly suitable for storing and maintainingpharmaceutical compositions having phosphate or citrate based buffers.According to the present invention, it has been discovered thatphosphate or citrate based buffers serve to increase delamination ofglass compositions. According in particular embodiments, thepharmaceutical composition includes a buffer comprising a salt ofcitrate, e.g., sodium citrate, or SSC. In other embodiments, thepharmaceutical composition includes a buffer comprising a salt ofphosphate, e.g., mono or disodium phosphate.

In additional embodiments, the pharmaceutical containers of the presentinvention are particularly suitable for storing and maintaining activepharmaceutical ingredient that needs to be subsequently formulated. Inother embodiments, the pharmaceutical containers of the presentinvention are particularly suitable for storing and maintaining alyophilized pharmaceutical composition or active pharmaceuticalingredient that requires reconstitution, for example, by addition ofsaline.

Assaying for Delamination of Pharmaceutical Containers

As noted above, delamination may result in the release of silica-richglass flakes into a solution contained within the glass container afterextended exposure to the solution. Accordingly, the resistance todelamination may be characterized by the number of glass particulatespresent in a solution contained within the glass container afterexposure to the solution under specific conditions. In order to assessthe long-term resistance of the glass container to delamination, anaccelerated delamination test was utilized. The test consisted ofwashing the glass container at room temperature for 1 minute anddepyrogenating the container at about 320° C. for 1 hour. Thereafter asolution of 20 mM glycine with a pH of 10 in water is placed in theglass container to 80-90% fill, the glass container is closed, andrapidly heated to 100° C. and then heated from 100° C. to 121° C. at aramp rate of 1 deg/min at a pressure of 2 atmospheres. The glasscontainer and solution are held at this temperature for 60 minutes,cooled to room temperature at a rate of 0.5 deg./min and the heatingcycle and hold are repeated. The glass container is then heated to 50°C. and held for two days for elevated temperature conditioning. Afterheating, the glass container is dropped from a distance of at least 18″onto a firm surface, such as a laminated tile floor, to dislodge anyflakes or particles that are weakly adhered to the inner surface of theglass container.

Thereafter, the solution contained in the glass container is analyzed todetermine the number of glass particles present per liter of solution.Specifically, the solution from the glass container is directly pouredonto the center of a Millipore Isopore Membrane filter (Millipore#ATTP02500 held in an assembly with parts #AP1002500 and #M000025A0)attached to vacuum suction to draw the solution through the filterwithin 10-15 seconds. Particulate flakes are then counted bydifferential interference contrast microscopy (DIC) in the reflectionmode as described in “Differential interference contrast (DIC)microscopy and modulation contrast microscopy” from Fundamentals oflight microscopy and digital imaging. New York: Wiley-Liss, pp 153-168.The field of view is set to approximately 1.5 mm×1.5 mm and particleslarger than 50 microns are counted manually. There are 9 suchmeasurements made in the center of each filter membrane in a 3×3 patternwith no overlap between images. A minimum of 100 mL of solution istested. As such, the solution from a plurality of small containers maybe pooled to bring the total amount of solution to 100 mL. If thecontainers contain more than 10 mL of solution, the entire amount ofsolution from the container is examined for the presence of particles.For containers having a volume greater than 10 mL, the test is repeatedfor a trial of 10 containers formed from the same glass compositionunder the same processing conditions and the result of the particlecount is averaged for the 10 containers to determine an average particlecount. Alternatively, in the case of small containers, the test isrepeated for a trial of 10 sets of 10 mL of solution, each of which isanalyzed and the particle count averaged over the 10 sets to determinean average particle count. Averaging the particle count over multiplecontainers accounts for potential variations in the delaminationbehavior of individual containers. Table 7 summarizes some non-limitingexamples of sample volumes and numbers of containers for testing isshown below:

TABLE 7 Table of Exemplary Test Specimens Nominal Total Vial Vial MaxMinimum Number of solution Capacity Volume Solution per Vials in aNumber of Tested (mL) (mL) Vial (mL) Trial Trials (mL) 2 4 3.2 4 10 1283.5 7 5.6 2 10 112 4 6 4.8 3 10 144 5 10 8 2 10 160 6 10 8 2 10 160 811.5 9.2 2 10 184 10 13.5 10.8 1 10 108 20 26 20.8 1 10 208 30 37.5 30 110 300 50 63 50.4 1 10 504

It should be understood that the aforementioned test is used to identifyparticles which are shed from the interior wall(s) of the glasscontainer due to delamination and not tramp particles present in thecontainer from forming processes or particles which precipitate from thesolution enclosed in the glass container as a result of reactionsbetween the solution and the glass. Specifically, delamination particlesmay be differentiated from tramp glass particles based on the aspectratio of the particle (i.e., the ratio of the width of the particle tothe thickness of the particle). Delamination produces particulate flakesor lamellae which are irregularly shaped and are typically >50 μm indiameter but often >200 μm. The thickness of the flakes is usuallygreater than about 100 nm and may be as large as about 1 μm. Thus, theminimum aspect ratio of the flakes is typically >50. The aspect ratiomay be greater than 100 and sometimes greater than 1000. Particlesresulting from delamination processes generally have an aspect ratiowhich is generally greater than about 50. In contrast, tramp glassparticles will generally have a low aspect ratio which is less thanabout 3. Accordingly, particles resulting from delamination may bedifferentiated from tramp particles based on aspect ratio duringobservation with the microscope. Validation results can be accomplishedby evaluating the heel region of the tested containers. Uponobservation, evidence of skin corrosion/pitting/flake removal, asdescribed in “Nondestructive Detection of Glass Vial Inner SurfaceMorphology with Differential Interference Contrast Microscopy” fromJournal of Pharmaceutical Sciences 101(4), 2012, pages 1378-1384, isnoted.

In the embodiments described herein, glass containers which average lessthan 3 glass particles with a minimum width of 50 μm and an aspect ratioof greater than 50 per trial following accelerated delamination testingare considered “delamination resistant.” In the embodiments describedherein, glass containers which average less than 2 glass particles witha minimum width of 50 μm and an aspect ratio of greater than 50 pertrial following accelerated delamination testing are considered“delamination-stable.” In the embodiments described herein, glasscontainers which average less than 1 glass particle with a minimum widthof 50 μm and an aspect ratio of greater than 50 per trial followingaccelerated delamination testing are considered “delamination-proof.” Inthe embodiments described herein, glass containers which have 0 glassparticles with a minimum width of 50 μm and an aspect ratio of greaterthan 50 per trial following accelerated delamination testing areconsidered “delamination-free”.

Assessing Stability of Pharmaceutical Compositions

As set forth above, any of a variety of active pharmaceuticalingredients can be incorporated within the claimed pharmaceuticalcontainer including, for example, a small molecule, a polypeptidemimetic, a biologic, an antisense RNA, a small interfering RNA (siRNA),etc. These active ingredients degrade in varying manners and, thus,assessing the stability thereof in the pharmaceutical containers of thepresent invention requires different techniques.

Depending on the nature of the active pharmaceutical ingredient, thestability, maintenance and/or continued efficacy of the pharmaceuticalcompositions contained within the delamination resistant pharmaceuticalcontainers of the present invention can be evaluated as follows.

Biologics API are often susceptible to degradation and/or inactivationarising from various factors, including pH, temperature, temperaturecycling, light, humidity, etc. Biologics API are further susceptible todegradation, inactivation or loss, arising from interaction of thepharmaceutical composition with the pharmaceutical container, ordelaminants leeching therefrom. For example, biologics may undergophysical degradation which may render the resulting pharmaceuticalcomposition inactive, toxic or insufficient to achieve the desiredeffect. Alternatively, or in addition, biologics may undergo structuralor conformational changes that can alter the activity of the API, withor without degradation. For example, proteins may undergo unfoldingwhich can result in effective loss and inactivity of the API.Alternatively, or in addition, biologics may adhere to the surface ofthe container, thereby rendering the API administered to the subjectinsufficient to achieve the desired effect, e.g., therapeutic effect.

(i) General Methods for Investigation of Biologic Compound Degradation

Depending on the size and complexity of the biologic, methods foranalysis of degradation of non-biologic, small molecule API may beapplied to biologics. For example, peptides and nucleic acids can beanalyzed using any of a number of chromatography and spectrometrytechniques applicable to small molecules to determine the size of themolecules, either with or without protease or nuclease digestion.However, as proper secondary and tertiary structures are required forthe activity of biologics, particularly protein biologics, confirmationof molecular weight is insufficient to confirm activity of biologics.Protein biologics containing complex post-translational modifications,e.g., glycosylation, are less amenable to analysis using chromatographyand spectrometry. Moreover, complex biologics, e.g., vaccines which caninclude complex peptide mixtures, attenuated or killed viruses, orkilled cells, are not amenable to analysis by most chromatography orspectrometry methods.

(ii) In Vitro Functional Assays for Investigation of Compound Stability

One or more in vitro assays, optionally in combination with one or morein vivo assays, can be used to assess the stability and activity of theAPI. Functional assays to determine API stability can be selected basedon the structural class of the API and the function of the API.Exemplary assays are provided below to confirm the activity of the APIafter stability and/or stress testing. It is understood that assaysshould be performed with the appropriate controls (e.g., vehiclecontrols, control API not subject to stress or stability testing) with asufficient number of dilutions and replicate samples to provide datawith sufficient statistical significance to detect changes in activityof 10% or less, preferably 5% or less, 4% or less, more preferably 3% orless, 2% or less, or 1% or less, as desired. Such considerations in theart are well understood.

For example, antibody based therapeutics, regardless of the disease orcondition to be treated, can be assayed for stability and activity usingassays that require specific binding of the antibody to its cognateantigen, e.g., ELISA. The antigen used in the ELISA should have theappropriate conformational structure as would be found in vivo. Antibodybased API are used, for example, for the treatment of cancer andinflammatory diseases including autoimmune diseases.

ELISA assays to assay the concentration of a protein biologic API arecommercially available from a number of sources, e.g., R&D Systems, BDBiosciences, AbCam, Pierce, Invitrogen.

API are frequently targeted to receptors, particularly cell surfacereceptors. Receptor binding assays can be used to assess the activity ofsuch agents. API that bind cell surface receptors can be agonists,antagonists or allosteric modulators. API that bind cell surfacereceptors need not bind the same location as the native ligand tomodulate, for example, inhibit or enhance, signaling through thereceptor. Depending on the activity of the API, an appropriate assay canbe selected, e.g., assay for stimulation of receptor signaling when theAPI is a receptor agonist; and inhibition assay in which binding of anagonist, e.g., inhibition of activation by a receptor agonist by theAPI. Such assays can be used regardless of the disease(s) orcondition(s) to be treated with the API. Modulation of cellularactivity, e.g., cell proliferation, apoptosis, cell migration,modulation of expression of genes or proteins, differentiation, tubeformation, etc. is assayed using routine methods. In other assaymethods, a reporter construct is used to indicate activation of thereceptor. Such methods are routine in the art. APIs that bind to cellsurface receptors are used, for example, as anti-cancer agents,anti-diabetic agents, anti-inflammatory agents for the treatment ofinflammatory mediated diseases including autoimmune disorders,anti-angiogenic agents, anti-cholinergic agents, bone calciumregulators, muscle and vascular tension regulators, and psychoactiveagents.

Modulators of cell proliferation can be assayed for activity using acell proliferation assays. For example, cell proliferation is inducedusing anti-anemic agents or stimulators of hematopoietic cell growth.Anti-proliferative agents, e.g., cytotoxic agents, anti-neoplasticagents, chemotherapeutic agents, cytostatic agents, antibiotic agents,are used to inhibit growth of various cell types. Some anti-inflammatoryagents also act by inhibiting proliferation of immune cells, e.g., blastcells. In proliferation assays, replicate wells containing the samenumber of cells are cultured in the presence of the API. The effect ofthe API is assessed using, for example, microscopy or fluorescenceactivated cell sorting (FACS) to determine if the number of cells in thesample increased or decreased in response to the presence of the API. Itis understood that the cell type selected for the proliferation assay isdependent on the specific API to be tested.

Modulators of angiogenesis can be assayed using cell migration and/ortube formation assays. For cell migration assays, human vascularendothelial cells (HUVECs) are cultured in the presence of the API intranswell devices. Migration of cells through the device at the desiredtime intervals is assessed. Alternatively, 3-dimensional HUVECs culturesin MATRIGEL can be assessed for tube formation. Anti-angiogenic agentsare used, for example, for the treatment of cancer, maculardegeneration, and diabetic retinopathy.

Anti-inflammatory API can be assayed for their effects on immune cellstimulation as determined, for example, by modulation of one or more ofcytokine expression and secretion, antigen presentation, migration inresponse to cytokine or chemokine stimulation, and immune cellproliferation. In such assays, immune cells are cultured in the presenceof the API and changes in immune cell activity are determined usingroutine methods in the art, e.g., ELISA and cell imaging and counting.

Anti-diabetic API can be assayed for their effects on insulin signaling,including insulin signaling in response to modulated glucose levels, andinsulin secretion. Insulin signaling can be assessed by assessing kinaseactivation in response to exposure to insulin and/or modulation ofglucose levels. Insulin secretion can be assessed by ELISA assay.

Modulators of blood clotting, i.e., fibrinolytics, anti-fibrinolytics,and anti-coagulants, can be assayed for their effects using an INR assayon serum by measuring prothrombin time to determine a prothrombin ratio.Time to formation of a clot is assayed in the presence or absence of theAPI.

Modulators of muscle or vascular tone can be assayed for their effectsusing vascular or muscle explants. The tissue can be placed in a caliperfor detection of changes in length and/or tension. Whole coronaryexplants can be used to assess the activity of API on heart. The tissueis contacted with the API, and optionally agents to alter vascular tone(e.g., K⁺, Ca⁺⁺). The effects of the API on length and/or tension of thevasculature or muscle is assessed.

Psychoactive agents can act by modulation of neurotransmitter releaseand/or recycling. Neuronal cells can be incubated in the presence of anAPI and stimulated to release neurotransmitters. Neurotransmitter levelscan be assessed in the culture medium collected at defined time pointsto detect alterations in the level of neurotransmitter present in themedia. Neurotransmitters can be detected, for example, using ELISA,LC/MS/MS, or by preloading the vesicles with radioactiveneurotransmitters to facilitate detection.

(iii) In Vivo Assays for Investigation of Compound Stability

In addition to in vitro testing for compound stability, API can also betested in vivo to confirm the stability of the API after storage and/orstress testing. For example, some API are not amenable to testing usingin vitro assays due to the complexity of the disease state or thecomplexity of the response required. For example, psychoactive agents,e.g., antipsychotic agents, anti-depressant agents, nootropic agents,immunosuppressant agents, vasotherapeutic agents, muscular dystrophyagents, central nervous system modulating agents, antispasmodic agents,bone calcium regenerating agents, anti-rheumatic agents,anti-hyperlipidemic agents, hematopoietic proliferation agents, growthfactors, vaccine agents, and imaging agents, may not be amenable to fullfunctional characterization using in vitro models. Moreover, for someagents, factors that may not alter in vitro activity may alter activityin vivo, e.g., antibody variable domains may be sufficient to blocksignaling through a receptor, but the Fc domains may be required forefficacy in the treatment of disease. Further, changes in stability mayresult in changes in pharmacokinetic properties of an API (e.g.,half-life, serum protein binding, tissue distribution, CNSpermeability). Finally, changes in stability may result in thegeneration of toxic degradation or reaction products that would not bedetected in vivo. Therefore, confirmation of pharmacokinetic andpharmacodynamic properties and toxicity in vivo is useful in conjunctionwith stability and stress testing.

(iv) Pharmacokinetic Assays

Pharmacokinetics includes the study of the mechanisms of absorption anddistribution of an administered drug, the rate at which a drug actionbegins and the duration of the effect, the chemical changes of thesubstance in the body (e.g. by metabolic enzymes such as CYP or UGTenzymes) and the effects and routes of excretion of the metabolites ofthe drug. Pharmacokinetics is divided into several areas including theextent and rate of absorption, distribution, metabolism and excretion.This is commonly referred to as the ADME scheme:

-   -   Absorption—the process of a substance entering the blood        circulation.    -   Distribution—the dispersion or dissemination of substances        throughout the fluids and tissues of the body.    -   Metabolism (or Biotransformation)—the irreversible        transformation of parent compounds into daughter metabolites.    -   Excretion—the removal of the substances from the body. In rare        cases, some drugs irreversibly accumulate in body tissue.    -   Elimination is the result of metabolism and excretion.

Pharmacokinetics describes how the body affects a specific drug afteradministration. Pharmacokinetic properties of drugs may be affected byelements such as the site of administration and the dose of administereddrug, which may affect the absorption rate. Such factors cannot be fullyassessed using in vitro models.

The specific pharmacokinetic properties to be assessed for a specificAPI in stability testing will depend, for example, on the specific APIto be tested. In vitro pharmacokinetic assays can include assays of drugmetabolism by isolated enzymes or by cells in culture. However,pharmacokinetic analysis typically requires analysis in vivo.

As pharmacokinetics are not concerned with the activity of the drug, butinstead with the absorption, distribution, metabolism, and excretion ofthe drug, assays can be performed in normal subjects, rather thansubjects suffering from a disease or condition for which the API istypically administered, by administration of a single dose of the API tothe subject. However, if the subject to be treated with the API has acondition that would alter the metabolism or excretion of the API, e.g.,liver disease, kidney disease, testing of the API in an appropriatedisease model may be useful. Depending on the half-life of the compound,samples (e.g., serum, urine, stool) are collected at predetermined timepoints for at least two, preferably three half-lives of the API, andanalyzed for the presence of the API and metabolic products of the API.At the end of the study, organs are harvested and analyzed for thepresence of the API and metabolic products of the API. Thepharmacokinetic properties of the API subjected to stability and/orstress testing are compared to API not subjected to stability or stresstesting and other appropriate controls (e.g., vehicle control). Changesin pharmacokinetic properties as a result of stability and/or stresstesting are determined.

(v) Pharmacodynamic Assays

Pharmacodynamics includes the study of the biochemical and physiologicaleffects of drugs on the body or on microorganisms or parasites within oron the body and the mechanisms of drug action and the relationshipbetween drug concentration and effect. Due to the complex nature of manydisease states and the actions of many API, the API should be tested invivo to confirm the desired activity of the agent. Mouse models for alarge variety of disease states are known and commercially available(see, e.g.,jaxmice.jax.org/query/f?p=205:1:989373419139701::::P1_ADV:1). A numberof induced models of disease are also known. Agents can be tested on theappropriate animal model to demonstrate stability and efficacy of theAPI on modulating the disease state.

(vi) Specific Immune Response Assay

Vaccines produce complex immune responses that are best assessed invivo. The specific potency assay for a vaccine depends, at least inpart, on the specific vaccine type. The most accurate predictions arebased on mathematical modeling of biologically relevantstability-indicating parameters. For complex vaccines, e.g., whole cellvaccines, whole virus vaccines, complex mixtures of antigens,characterization of each component biochemically may be difficult, ifnot impossible. For example, when using a live, attenuated virusvaccine, the number of plaque forming units (e.g., mumps, measles,rubella, smallpox) or colony forming units (e.g., S. typhi, TY21a) aredetermined to confirm potency after storage. Chemical and physicalcharacterization (e.g., polysaccharide and polysaccharide-proteinconjugate vaccines) is performed to confirm the stability and activityof the vaccine. Serological response in animals to inactivated toxinsand/or animal protection against challenge (e.g., rabies, anthrax,diphtheria, tetanus) is performed to confirm activity of vaccines of anytype, particularly when the activity of the antigen has beeninactivated. In vivo testing of vaccines subjected to stability and/orstress testing is performed by administering the vaccine to a subjectusing the appropriate immunization protocol for the vaccine, anddetermining the immune response by detection of specific immune cellsthat respond to stimulation with the antigen or pathogen, detection ofantibodies against the antigen or pathogen, or protection in an immunechallenge. Such methods are well known in the art. Vaccines include, butare not limited to, meningococcal B vaccine, hepatitis A and B vaccines,human papillomavirus vaccine, influenza vaccine, herpes zoster vaccine,and pneumococcal vaccine.

(vii) Toxicity Assays

Degradation of API can result in in the formation of toxic agents.Toxicity assays include the administration of doses, typically farhigher than would be used for therapeutic applications, to detect thepresence of toxic products in the API. Toxicity assays can be performedin vitro and in vivo and are frequently single, high dose experiments.After administration of the compound, in addition to viability, organsare harvested and analyzed for any indication of toxicity, especiallyorgans involved with clearance of API, e.g., liver, kidneys, and thosefor which damage could be catastrophic, e.g., heart, brain. Thetoxicologic properties of the API subjected to stability and/or stresstesting are compared to API not subjected to stability or stress testingand other appropriate controls (e.g., vehicle control). Changes intoxicologic properties as a result of stability and/or stress testingare determined.

In accordance with present invention, the degradation, alteration ordepletion of API contained within a delamination resistantpharmaceutical container of the present invention can be assessed by avariety of physical techniques. Indeed, in various aspects of theinvention, the stability and degradation caused by the interaction ofAPI with the container or delaminants thereof, or changes inconcentration or amount of the API in a container can be assessed usingtechniques as follows. Such methods include, e.g., X-Ray Diffraction(XRPD), Thermal Analysis (such as Differential Scanning calorimetry(DSC), Thermogravimetry (TG) and Hot-Stage Microscopy (HSM),chromatography methods (such as High-Performance Liquid Chromatography(HPLC), Column Chromatography (CC), Gas Chromatography (GC), Thin-LayerChromatography (TLC), and Super Critical Phase Chromatograph (SFC)),Mass Spectroscopy (MS), Capillary Electrophoresis (CE), AtomicSpectroscopy (AS), vibrational spectroscopy (such as InfraredSpectroscopy (IR)), Luminescence Spectroscopy (LS), and Nuclear MagneticResonance Spectroscopy (NMR).

In the case of pharmaceutical formulations where the API is not insolution or needs to be reconstituted into a different medium, XRPD maybe a method for analyzing degradation. In ideal cases, every possiblecrystalline orientation is represented equally in a non-liquid sample.

Powder diffraction data is usually presented as a diffractogram in whichthe diffracted intensity I is shown as function either of the scatteringangle 20 or as a function of the scattering vector q. The lattervariable has the advantage that the diffractogram no longer depends onthe value of the wavelength λ. Relative to other methods of analysis,powder diffraction allows for rapid, non-destructive analysis ofmulti-component mixtures without the need for extensive samplepreparation. Deteriorations of an API may be analyzed using this method,e.g., by comparing the diffraction pattern of the API to a knownstandard of the API prior to packaging.

Thermal methods of analysis may include, e.g., differential scanningcalorimetry (DSC), thermogravimetry (TG), and hot-stage microscopy(HSM). All three methods provide information upon heating the sample.Depending on the information required, heating can be static or dynamicin nature.

Differential scanning calorimetry monitors the energy required tomaintain the sample and a reference at the same temperature as they areheated. A plot of heat flow (W/g or J/g) versus temperature is obtained.The area under a DSC peak is directly proportional to the heat absorbedor released and integration of the peak results in the heat oftransition.

Thermogravimetry (TG) measures the weight change of a sample as afunction of temperature. A total volatile content of the sample isobtained, but no information on the identity of the evolved gas isprovided. The evolved gas must be identified by other methods, such asgas chromatography, Karl Fisher titration (specifically to measurewater), TG-mass spectroscopy, or TG-infrared spectroscopy. Thetemperature of the volatilization and the presence of steps in the TGcurve can provide information on how tightly water or solvent is held inthe lattice. If the temperature of the TG volatilization is similar toan endothermic peak in the DSC, the DSC peak is likely due or partiallydue to volatilization. It may be necessary to utilize multipletechniques to determine if more than one thermal event is responsiblefor a given DSC peak.

Hot-Stage Microscopy (HSM) is a technique that supplements DSC and TG.Events observed by DSC and/or TG can be readily characterized by HSM.Melting, gas evolution, and solid-solid transformations can bevisualized, providing the most straightforward means of identifyingthermal events. Thermal analysis can be used to determine the meltingpoints, recrystallizations, solid-state transformations, decompositions,and volatile contents of pharmaceutical materials.

Other methods to analyze degradation or alteration of API and excipientsare infrared (IR) and Raman spectroscopy. These techniques are sensitiveto the structure, conformation, and environment of organic compounds.Infrared spectroscopy is based on the conversion of IR radiation intomolecular vibrations. For a vibration to be IR-active, it must involve achanging molecular dipole (asymmetric mode). For example, vibration of adipolar carbonyl group is detectable by IR spectroscopy. Whereas IR hasbeen traditionally used as an aid in structure elucidation, vibrationalchanges also serve as probes of intermolecular interactions in solidmaterials.

Raman spectroscopy is based on the inelastic scattering of laserradiation with loss of vibrational energy by a sample. A vibrationalmode is Raman active when there is a change in the polarizability duringthe vibration. Symmetric modes tend to be Raman-active. For example,vibrations about bonds between the same atom, such as in alkynes, can beobserved by Raman spectroscopy.

NMR spectroscopy probes atomic environments based on the differentresonance frequencies exhibited by nuclei in a strong magnetic field.Many different nuclei are observable by the NMR technique, but those ofhydrogen and carbon atoms are most frequently studied. Solid-state NMRmeasurements are extremely useful for characterizing the crystal formsof pharmaceutical solids. Nuclei that are typically analyzed with thistechnique include those of 13C, 31P, 15N, 25Mg, and 23Na.

Chromatography is a general term applied to a wide variety of separationtechniques based on the sample partitioning between a moving phase,which can be a gas, liquid, or supercritical fluid, and a stationaryphase, which may be either a liquid or a solid. Generally, the crux ofchromatography lies in the highly selective chemical interactions thatoccur in both the mobile and stationary phases. For example, dependingon the API and the separation required, one or more of absorption,ion-exchange, size-exclusion, bonded phase, reverse, or normal phasestationary phases may be employed.

Mass spectrometry (MS) is an analytical technique that works by ionizingchemical compounds to generate charged molecules or molecule fragmentsand measuring their mass-to-charge ratios. Based on this analysismethod, one can determine, e.g., the isotopic composition of elements inan API and determine the structure of the API by observing itsfragmentation pattern.

It would be understood that the foregoing methods do not represent acomprehensive list of means by which one can analyze possibledeteriorations, alterations, or concentrations of certain APIs.Therefore, it would be understood that other methods for determining thephysical amounts and/or characteristics of an API may be employed.Additional methods may include, but are not limited to, e.g., CapillaryElectrophoresis (CE), Atomic Spectroscopy (AS), and LuminescenceSpectroscopy (LS).

EXAMPLES

The embodiments of the delamination resistant pharmaceutical containersdescribed herein will be further clarified by the following examples.

Example 1

Six exemplary inventive glass compositions (compositions A-F) wereprepared. The specific compositions of each exemplary glass compositionare reported below in Table 8. Multiple samples of each exemplary glasscomposition were produced. One set of samples of each composition wasion exchanged in a molten salt bath of 100% KNO₃ at a temperature of450° C. for at least 5 hours to induce a compressive layer in thesurface of the sample. The compressive layer had a surface compressivestress of at least 500 MPa and a depth of layer of at least 45 μm.

The chemical durability of each exemplary glass composition was thendetermined utilizing the DIN 12116 standard, the ISO 695 standard, andthe ISO 720 standard described above. Specifically, non-ion exchangedtest samples of each exemplary glass composition were subjected totesting according to one of the DIN 12116 standard, the ISO 695standard, or the ISO 720 standard to determine the acid resistance, thebase resistance or the hydrolytic resistance of the test sample,respectively. The hydrolytic resistance of the ion exchanged samples ofeach exemplary composition was determined according to the ISO 720standard. The average results of all samples tested are reported belowin Table 8.

As shown in Table 8, exemplary glass compositions A-F all demonstrated aglass mass loss of less than 5 mg/dm² and greater than 1 mg/dm²following testing according to the DIN 12116 standard with exemplaryglass composition E having the lowest glass mass loss at 1.2 mg/dm².Accordingly, each of the exemplary glass compositions were classified inat least class S3 of the DIN 12116 standard, with exemplary glasscomposition E classified in class S2. Based on these test results, it isbelieved that the acid resistance of the glass samples improves withincreased SiO₂ content.

Further, exemplary glass compositions A-F all demonstrated a glass massloss of less than 80 mg/dm² following testing according to the ISO 695standard with exemplary glass composition A having the lowest glass massloss at 60 mg/dm². Accordingly, each of the exemplary glass compositionswere classified in at least class A2 of the ISO 695 standard, withexemplary glass compositions A, B, D and F classified in class A1. Ingeneral, compositions with higher silica content exhibited lower baseresistance and compositions with higher alkali/alkaline earth contentexhibited greater base resistance.

Table 8 also shows that the non-ion exchanged test samples of exemplaryglass compositions A-F all demonstrated a hydrolytic resistance of atleast Type HGA2 following testing according to the ISO 720 standard withexemplary glass compositions C-F having a hydrolytic resistance of TypeHGA1. The hydrolytic resistance of exemplary glass compositions C-F isbelieved to be due to higher amounts of SiO₂ and the lower amounts ofNa₂O in the glass compositions relative to exemplary glass compositionsA and B.

Moreover, the ion exchanged test samples of exemplary glass compositionsB-F demonstrated lower amounts of extracted Na₂O per gram of glass thanthe non-ion exchanged test samples of the same exemplary glasscompositions following testing according to the ISO 720 standard.

TABLE 8 Composition and Properties of Exemplary Glass CompositionsComposition in mole % A B C D E F SiO₂ 70.8 72.8 74.8 76.8 76.8 77.4Al₂O₃ 7.5 7 6.5 6 6 7 Na₂O 13.7 12.7 11.7 10.7 11.6 10 K₂O 1 1 1 1 0.10.1 MgO 6.3 5.8 5.3 4.8 4.8 4.8 CaO 0.5 0.5 0.5 0.5 0.5 0.5 SnO₂ 0.2 0.20.2 0.2 0.2 0.2 DIN 12116 3.2 2.0 1.7 1.6 1.2 1.7 (mg/dm²)classification S3 S3 S3 S3 S2 S3 ISO 695 60.7 65.4 77.9 71.5 76.5 62.4(mg/dm²) classification A1 A1 A2 A1 A2 A1 ISO 720 100.7 87.0 54.8 57.550.7 37.7 (ug Na₂O/ g glass) classification HGA2 HGA2 HGA1 HGA1 HGA1HGA1 ISO 720 60.3 51.9 39.0 30.1 32.9 23.3 (with IX) (ug Na₂O/ g glass)classification HGA1 HGA1 HGA1 HGA1 HGA1 HGA1

Example 2

Three exemplary inventive glass compositions (compositions G-I) andthree comparative glass compositions (compositions 1-3) were prepared.The ratio of alkali oxides to alumina (i.e., Y:X) was varied in each ofthe compositions in order to assess the effect of this ratio on variousproperties of the resultant glass melt and glass. The specificcompositions of each of the exemplary inventive glass compositions andthe comparative glass compositions are reported in Table 9. The strainpoint, anneal point, and softening point of melts formed from each ofthe glass compositions were determined and are reported in Table 2. Inaddition, the coefficient of thermal expansion (CTE), density, andstress optic coefficient (SOC) of the resultant glasses were alsodetermined and are reported in Table 9. The hydrolytic resistance ofglass samples formed from each exemplary inventive glass composition andeach comparative glass composition was determined according to the ISO720 Standard both before ion exchange and after ion exchange in a moltensalt bath of 100% KNO₃ at 450° C. for 5 hours. For those samples thatwere ion exchanged, the compressive stress was determined with afundamental stress meter (FSM) instrument, with the compressive stressvalue based on the measured stress optical coefficient (SOC). The FSMinstrument couples light into and out of the birefringent glass surface.The measured birefringence is then related to stress through a materialconstant, the stress-optic or photoelastic coefficient (SOC or PEC) andtwo parameters are obtained: the maximum surface compressive stress (CS)and the exchanged depth of layer (DOL). The diffusivity of the alkaliions in the glass and the change in stress per square root of time werealso determined

TABLE 9 Glass properties as a function of alkali to alumina ratioComposition Mole % G H I 1 2 3 SiO₂ 76.965 76.852 76.962 76.919 76.96077.156 Al₂O₃ 5.943 6.974 7.958 8.950 4.977 3.997 Na₂O 11.427 10.4739.451 8.468 12.393 13.277 K₂O 0.101 0.100 0.102 0.105 0.100 0.100 MgO4.842 4.878 4.802 4.836 4.852 4.757 CaO 0.474 0.478 0.481 0.480 0.4680.462 SnO₂ 0.198 0.195 0.197 0.197 0.196 0.196 Strain (° C.) 578 616 654683 548 518 Anneal (° C.) 633 674 716 745 600 567 Softening (° C.) 892946 1003 1042 846 798 Expansion (10⁻⁷ K⁻¹) 67.3 64.3 59.3 55.1 71.8 74.6Density (g/cm³) 2.388 2.384 2.381 2.382 2.392 2.396 SOC (nm/mm/Mpa)3.127 3.181 3.195 3.232 3.066 3.038 ISO720 (non-IX) 88.4 60.9 47.3 38.4117.1 208.1 ISO720 (IX450° C.-5 hr) 25.3 26 20.5 17.8 57.5 102.5R₂O/Al₂O₃ 1.940 1.516 1.200 0.958 2.510 3.347 CS@t = 0 (MPa) 708 743 738655 623 502 CS/√t (MPa/hr^(1/2)) −35 −24 −14 −7 −44 −37 D (μm²/hr) 52.053.2 50.3 45.1 51.1 52.4

The data in Table 9 indicates that the alkali to alumina ratio Y:Xinfluences the melting behavior, hydrolytic resistance, and thecompressive stress obtainable through ion exchange strengthening. Inparticular, FIG. 1 graphically depicts the strain point, anneal point,and softening point as a function of Y:X ratio for the glasscompositions of Table 9. FIG. 1 demonstrates that, as the ratio of Y:Xdecreases below 0.9, the strain point, anneal point, and softening pointof the glass rapidly increase. Accordingly, to obtain a glass which isreadily meltable and formable, the ratio Y:X should be greater than orequal to 0.9 or even greater than or equal to 1.

Further, the data in Table 2 indicates that the diffusivity of the glasscompositions generally decreases with the ratio of Y:X. Accordingly, toachieve glasses can be rapidly ion exchanged in order to reduce processtimes (and costs) the ratio of Y:X should be greater than or equal to0.9 or even greater than or equal to 1.

Moreover, FIG. 2 indicates that for a given ion exchange time and ionexchange temperature, the maximum compressive stresses are obtained whenthe ratio of Y:X is greater than or equal to about 0.9, or even greaterthan or equal to about 1, and less than or equal to about 2,specifically greater than or equal to about 1.3 and less than or equalto about 2.0. Accordingly, the maximum improvement in the load bearingstrength of the glass can be obtained when the ratio of Y:X is greaterthan about 1 and less than or equal to about 2. It is generallyunderstood that the maximum stress achievable by ion exchange will decaywith increasing ion-exchange duration as indicated by the stress changerate (i.e., the measured compressive stress divided by the square rootof the ion exchange time). FIG. 2 generally shows that the stress changerate decreases as the ratio Y:X decreases.

FIG. 3 graphically depicts the hydrolytic resistance (y-axis) as afunction of the ratio Y:X (x-axis). As shown in FIG. 3, the hydrolyticresistance of the glasses generally improves as the ratio Y:X decreases.

Based on the foregoing it should be understood that glasses with goodmelt behavior, superior ion exchange performance, and superiorhydrolytic resistance can be achieved by maintaining the ratio Y:X inthe glass from greater than or equal to about 0.9, or even greater thanor equal to about 1, and less than or equal to about 2.

Example 3

Three exemplary inventive glass compositions (compositions J-L) andthree comparative glass compositions (compositions 4-6) were prepared.The concentration of MgO and CaO in the glass compositions was varied toproduce both MgO-rich compositions (i.e., compositions J-L and 4) andCaO-rich compositions (i.e., compositions 5-6). The relative amounts ofMgO and CaO were also varied such that the glass compositions haddifferent values for the ratio (CaO/(CaO+MgO)). The specificcompositions of each of the exemplary inventive glass compositions andthe comparative glass compositions are reported below in Table 10. Theproperties of each composition were determined as described above withrespect to Example 2.

TABLE 10 Glass properties as function of CaO content Composition Mole %J K L 4 5 6 SiO₂ 76.99 77.10 77.10 77.01 76.97 77.12 Al₂O₃ 5.98 5.975.96 5.96 5.97 5.98 Na₂O 11.38 11.33 11.37 11.38 11.40 11.34 K₂O 0.100.10 0.10 0.10 0.10 0.10 MgO 5.23 4.79 3.78 2.83 1.84 0.09 CaO 0.07 0.451.45 2.46 3.47 5.12 SnO₂ 0.20 0.19 0.19 0.19 0.19 0.19 Strain (° C.) 585579 568 562 566 561 Anneal (° C.) 641 634 620 612 611 610 Softening (°C.) 902 895 872 859 847 834 Expansion (10⁻⁷ K⁻¹) 67.9 67.1 68.1 68.869.4 70.1 Density (g/cm³) 2.384 2.387 2.394 2.402 2.41 2.42 SOCnm/mm/Mpa 3.12 3.08 3.04 3.06 3.04 3.01 ISO720 (non-IX) 83.2 83.9 86 8688.7 96.9 ISO720 (IX450° C.-5 hr) 29.1 28.4 33.2 37.3 40.1 Fraction ofRO as CaO 0.014 0.086 0.277 0.465 0.654 0.982 CS@t = 0 (MPa) 707 717 713689 693 676 CS/√t (MPa/hr^(1/2)) −36 −37 −39 −38 −43 −44 D (μm²/hr) 57.250.8 40.2 31.4 26.4 20.7

FIG. 4 graphically depicts the diffusivity D of the compositions listedin Table 10 as a function of the ratio (CaO/(CaO+MgO)). Specifically,FIG. 4 indicates that as the ratio (CaO/(CaO+MgO)) increases, thediffusivity of alkali ions in the resultant glass decreases therebydiminishing the ion exchange performance of the glass. This trend issupported by the data in Table 10 and FIG. 5. FIG. 5 graphically depictsthe maximum compressive stress and stress change rate (y-axes) as afunction of the ratio (CaO/(CaO+MgO)). FIG. 5 indicates that as theratio (CaO/(CaO+MgO)) increases, the maximum obtainable compressivestress decreases for a given ion exchange temperature and ion exchangetime. FIG. 5 also indicates that as the ratio (CaO/(CaO+MgO)) increases,the stress change rate increases (i.e., becomes more negative and lessdesirable).

Accordingly, based on the data in Table 10 and FIGS. 4 and 5, it shouldbe understood that glasses with higher diffusivities can be produced byminimizing the ratio (CaO/(CaO+MgO)). It has been determined thatglasses with suitable diffusivities can be produced when the(CaO/(CaO+MgO)) ratio is less than about 0.5. The diffusivity values ofthe glass when the (CaO/(CaO+MgO)) ratio is less than about 0.5decreases the ion exchange process times needed to achieve a givencompressive stress and depth of layer. Alternatively, glasses withhigher diffusivities due to the ratio (CaO/(CaO+MgO)) may be used toachieve a higher compressive stress and depth of layer for a given ionexchange temperature and ion exchange time.

Moreover, the data in Table 10 also indicates that decreasing the ratio(CaO/(CaO+MgO)) by increasing the MgO concentration generally improvesthe resistance of the glass to hydrolytic degradation as measured by theISO 720 standard.

Example 4

Three exemplary inventive glass compositions (compositions M-O) andthree comparative glass compositions (compositions 7-9) were prepared.The concentration of B₂O₃ in the glass compositions was varied from 0mol. % to about 4.6 mol. % such that the resultant glasses had differentvalues for the ratio B₂O₃/(R₂O−Al₂O₃). The specific compositions of eachof the exemplary inventive glass compositions and the comparative glasscompositions are reported below in Table 11. The properties of eachglass composition were determined as described above with respect toExamples 2 and 3.

TABLE 11 Glass properties as a function of B₂O₃ content Composition Mole% M N O 7 8 9 SiO₂ 76.860 76.778 76.396 74.780 73.843 72.782 Al₂O₃ 5.9645.948 5.919 5.793 5.720 5.867 B₂O₃ 0.000 0.214 0.777 2.840 4.443 4.636Na₂O 11.486 11.408 11.294 11.036 10.580 11.099 K₂O 0.101 0.100 0.1000.098 0.088 0.098 MgO 4.849 4.827 4.801 4.754 4.645 4.817 CaO 0.4920.480 0.475 0.463 0.453 0.465 SnO₂ 0.197 0.192 0.192 0.188 0.183 0.189Strain (° C.) 579 575 572 560 552 548 Anneal (° C.) 632 626 622 606 597590 Softening (° C.) 889 880 873 836 816 801 Expansion (10⁻⁷ K⁻¹) 68.367.4 67.4 65.8 64.1 67.3 Density (g/cm³) 2.388 2.389 2.390 2.394 2.3922.403 SOC (nm/mm/MPa) 3.13 3.12 3.13 3.17 3.21 3.18 ISO720 (non-IX) 86.378.8 68.5 64.4 52.7 54.1 ISO720 (IX450° C.-5 hr) 32.2 30.1 26 24.7 22.626.7 B₂O₃/(R₂O—Al₂O₃) 0.000 0.038 0.142 0.532 0.898 0.870 CS@t = 0 (MPa)703 714 722 701 686 734 CS/√t (MPa/hr^(1/2)) −38 −38 −38 −33 −32 −39 D(μm²/hr) 51.7 43.8 38.6 22.9 16.6 15.6

FIG. 6 graphically depicts the diffusivity D (y-axis) of the glasscompositions in Table 11 as a function of the ratio B₂O₃/(R₂O−Al₂O₃)(x-axis) for the glass compositions of Table 11. As shown in FIG. 6, thediffusivity of alkali ions in the glass generally decreases as the ratioB₂O₃/(R₂O−Al₂O₃) increases.

FIG. 7 graphically depicts the hydrolytic resistance according to theISO 720 standard (y-axis) as a function of the ratio B₂O₃/(R₂O−Al₂O₃)(x-axis) for the glass compositions of Table 11. As shown in FIG. 6, thehydrolytic resistance of the glass compositions generally improves asthe ratio B₂O₃/(R₂O−Al₂O₃) increases.

Based on FIGS. 6 and 7, it should be understood that minimizing theratio B₂O₃/(R₂O−Al₂O₃) improves the diffusivity of alkali ions in theglass thereby improving the ion exchange characteristics of the glass.Further, increasing the ratio B₂O₃/(R₂O−Al₂O₃) also generally improvesthe resistance of the glass to hydrolytic degradation. In addition, ithas been found that the resistance of the glass to degradation in acidicsolutions (as measured by the DIN 12116 standard) generally improveswith decreasing concentrations of B₂O₃. Accordingly, it has beendetermined that maintaining the ratio B₂O₃/(R₂O−Al₂O₃) to less than orequal to about 0.3 provides the glass with improved hydrolytic and acidresistances as well as providing for improved ion exchangecharacteristics.

It should now be understood that the glass compositions described hereinexhibit chemical durability as well as mechanical durability followingion exchange. These properties make the glass compositions well suitedfor use in various applications including, without limitation,pharmaceutical packaging materials.

Example 5 Determining the Presence and Amount of Glass Flakes inPharmaceutical Solutions

The resistance to delamination may be characterized by the number ofglass particulates present in a pharmaceutical solution contained withina glass container described herein after. In order to assess thelong-term resistance of the glass container to delamination, anaccelerated delamination test is utilized. The test consists of washingthe glass container at room temperature for 1 minute and depyrogenatingthe container at about 320° C. for 1 hour. Thereafter a pharmaceuticalsolution is placed in the glass container to 80-90% full, the glasscontainer is closed, and rapidly heated to, for example, 100° C. andthen heated from 100° C. to 121° C. at a ramp rate of 1 deg/min at apressure of 2 atmospheres. The glass container and solution are held atthis temperature for 60 minutes, cooled to room temperature at a rate of0.5 deg/min and the heating cycle and hold are repeated. The glasscontainer is then heated to 50° C. and held for two days for elevatedtemperature conditioning. After heating, the glass container is droppedfrom a distance of at least 18″ onto a firm surface, such as a laminatedtile floor, to dislodge any flakes or particles that are weakly adheredto the inner surface of the glass container.

Thereafter, the pharmaceutical solution contained in the glass containeris analyzed to determine the number of glass particles present per literof solution. Specifically, the solution from the glass container isdirectly poured onto the center of a Millipore Isopore Membrane filter(Millipore #ATTP02500 held in an assembly with parts #AP1002500 and#M000025A0) attached to vacuum suction to draw the solution through thefilter within 10-15 seconds. Particulate flakes are then counted bydifferential interference contrast microscopy (DIC) in the reflectionmode as described in “Differential interference contrast (DIC)microscopy and modulation contrast microscopy” from Fundamentals oflight microscopy and digital imaging. New York: Wiley-Liss, pp 153-168.The field of view is set to approximately 1.5 mm×1.5 mm and particleslarger than 50 microns are counted manually. There are 9 suchmeasurements made in the center of each filter membrane in a 3×3 patternwith no overlap between images. A minimum of 100 mL of solution istested. As such, the solution from a plurality of small containers maybe pooled to bring the total amount of solution to 100 mL. If thecontainers contain more than 10 mL of solution, the entire amount ofsolution from the container is examined for the presence of particles.For containers having a volume greater than 10 mL containers, the testis repeated for a trial of 10 containers formed from the same glasscomposition under the same processing conditions and the result of theparticle count is averaged for the 10 containers to determine an averageparticle count. Alternatively, in the case of small containers, the testis repeated for a trial of 10 sets of 10 mL of solution, each of whichis analyzed and the particle count averaged over the 10 sets todetermine an average particle count. Averaging the particle count overmultiple containers accounts for potential variations in thedelamination behavior of individual containers.

It should be understood that the aforementioned test is used to identifyparticles which are shed from the interior wall(s) of the glasscontainer due to delamination and not tramp particles present in thecontainer from forming processes. Specifically, delamination particleswill be differentiated from tramp glass particles based on the aspectratio of the particle (i.e., the ratio of the width of the particle tothe thickness of the particle). Delamination produces particulate flakesor lamellae which are irregularly shaped and are typically >50 μm indiameter but often >200 μm. The thickness of the flakes is usuallygreater than about 100 nm and may be as large as about 1 μm. Thus, theminimum aspect ratio of the flakes is typically >50. The aspect ratiomay be greater than 100 and sometimes greater than 1000. Particlesresulting from delamination processes generally have an aspect ratiowhich is generally greater than about 50. In contrast, tramp glassparticles will generally have a low aspect ratio which is less thanabout 3. Accordingly, particles resulting from delamination may bedifferentiated from tramp particles based on aspect ratio duringobservation with the microscope. Validation results can be accomplishedby evaluating the heel region of the tested containers. Uponobservation, evidence of skin corrosion/pitting/flake removal, asdescribed in “Nondestructive Detection of Glass Vial Inner SurfaceMorphology with Differential Interference Contrast Microscopy” fromJournal of Pharmaceutical Sciences 101(4), 2012, pages 1378-1384, isnoted.

Using this method, pharmaceutical compositions can be tested for thepresence of glass flakes and various compositions can be compared toeach other to assess the safety of various pharmaceutical compositions.

Example 6 Stability Testing of Pharmaceutical Compositions

Stability studies are part of the testing required by the FDA and otherregulatory agencies. Stability studies should include testing of thoseattributes of the API that are susceptible to change during storage andare likely to influence quality, safety, and/or efficacy. The testingshould cover, as appropriate, the physical, chemical, biological, andmicrobiological attributes of the API (e.g., small molecule or biologictherapeutic agent) in the container with the closure to be used forstorage of the agent. If the API is formulated as a liquid by themanufacturer, the final formulation should be assayed for stability. Ifthe API is formulated as an agent for reconstitution by the end userusing a solution provided by the manufacturer, both the API and thesolution for reconstitution are preferably tested for stability as theseparate packaged components (e.g., the API subjected to storagereconstituted with solution for reconstitution not subject to storage,API not subject to storage reconstituted with a solution subject tostorage, and both API and solution subject to storage). This isparticularly the case when the solution for reconstitution includes anactive agent (e.g., an adjuvant for reconstitution of a vaccine).

In general, a substance API should be evaluated under storage conditions(with appropriate tolerances) that test its thermal stability and, ifapplicable, its sensitivity to moisture. The storage conditions and thelengths of studies chosen should be sufficient to cover storage,shipment, and subsequent use.

API should be stored in the container(s) in which the API will beprovided to the end user (e.g., vials, ampules, syringes, injectabledevices). Stability testing methods provided herein refer to samplesbeing removed from the storage or stress conditions indicated. Removalof a sample preferably refers to removing an entire container from thestorage or stress conditions. Removal of a sample should not beunderstood as withdrawing a portion of the API from the container asremoval of a portion of the API from the container would result inchanges of fill volume, gas environment, etc. At the time of testing theAPI subject to stability and/or stress testing, portions of the samplessubject to stability and/or stress testing can be used for individualassays.

The long-term testing should cover a minimum of 12 months' duration onat least three primary batches at the time of submission and should becontinued for a period of time sufficient to cover the proposed retestperiod. Additional data accumulated during the assessment period of theregistration application should be submitted to the authorities ifrequested. Data from the accelerated storage condition and, ifappropriate, from the intermediate storage condition can be used toevaluate the effect of short-term excursions outside the label storageconditions (such as might occur during shipping).

Long-term, accelerated, and, where appropriate, intermediate storageconditions for API are detailed in the sections below. The general caseshould apply if the API is not specifically covered by a subsequentsection. It is understood that the time points for analysis indicated inthe table are suggested end points for analysis. Interim analysis can beperformed at shorter time points (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or 11 months). For API to be labeled as stable for storage for more than12 months, time points beyond 12 months can be assessed (e.g., 15, 18,21, 24 months). Alternative storage conditions can be used if justified.

TABLE 12 General Conditions for Stability Analysis Time points StudyStorage condition for analysis Long-term Long-term* 25° C. ± 2° C./ 12months  60% RH ± 5% RH or 30° C. ± 2° C./65% RH ± 5% RH Intermediate 30°C. ± 2° C./65% RH ± 5% RH 6 months Accelerated 40° C. ± 2° C./75% RH ±5% RH 6 months

TABLE 13 Conditions for Stability Analysis for Storage in a RefrigeratorMinimum time period covered by data at Study Storage conditionsubmission Long-term  5° C. ± 3° C. 12 months Accelerated 25° C. ± 2°C./60% RH ± 5% RH  6 months

TABLE 14 Conditions for Stability Analysis for Storage in a FreezerMinimum time period covered by data at Study Storage conditionsubmission Long-term −20° C. ± 5° C. 12 months

Storage condition for API intended to be stored in a freezer, testing ona single batch at an elevated temperature (e.g., 5° C.±3° C. or 25°C.±2° C.) for an appropriate time period should be conducted to addressthe effect of short-term excursions outside the proposed label storagecondition (e.g., stress during shipping or handling, e.g., increasedtemperature, multiple freeze-thaw cycles, storage in a non-uprightorientation, shaking, etc.).

The assays performed to assess stability of an API include assays tothat are used across most APIs to assess the physical properties of theAPI, e.g., degradation, pH, color, particulate formation, concentration,toxicity, etc. Assays to detect the general properties of the API arealso selected based on the chemical class of the agent, e.g.,denaturation and aggregation of protein based API. Assays to detect thepotency of the API, i.e., the ability of the API to achieve its intendedeffect as demonstrated by the quantitative measurement of an attributeindicative of the clinical effect as compared to an appropriate control,are selected based on the activity of the particular agent. For example,the biological activity of the API, e.g., enzyme inhibitor activity,cell killing activity, anti-inflammatory activity, coagulationmodulating activity, etc., is measured using in vitro and/or in vivoassays such as those provided herein. Pharmacokinetic and toxicologicalproperties of the API are also assessed using methods known in the art,such as those provided herein.

Example 7 Analysis of Adherence to Glass Vials

Changes in the surface of glass can result in changes in the adherenceof API to glass. The amount of agent in samples withdrawn from glassvials are tested at intervals to determine if the concentration of theAPI in solution changes over time. API are incubated in containers asdescribed in the stability testing and/or stress testing methodsprovided in Example 6. Preferably, the API is incubated both in standardglass vials with appropriate closures and glass vials such as thoseprovided herein. At the desired intervals, samples are removed andassayed to determine the concentration of the API in solution. Theconcentration of the API is determined using methods and controlsappropriate to the API. The concentration of the API is preferablydetermined in conjunction with at least one assay to confirm that theAPI, rather than degradation products of the API, is detected. In thecase of biologics in which the conformational structure of the biologicagent is essential to its function of the API, the assays forconcentration of the biologic are preferably preformed in conjunctionwith an assay to confirm the structure of the biologic (e.g., activityassay).

For example, in the cases of small molecule APIs, the amount of agentpresent is determined, for example, by mass spectrometry, optionally incombination with liquid chromatography, as appropriate, to separate theagent from any degradation products that may be present in the sample.

For protein based biologic APIs, the concentration of the API isdetermined, for example, using ELISA assay. Chromatography methods areused in conjunction with methods to determine protein concentration toconfirm that protein fragments or aggregates are not being detected bythe ELISA assay.

For nucleic acid biologic APIs, the concentration of the API isdetermined, for example, using quantitative PCR when the nucleic acidsare of sufficient length to permit detection by such methods.Chromatography methods are used to determine both the concentration andsize of nucleic acid based API.

For viral vaccine APIs, the concentration of the virus is determined,for example, using colony formation assays.

Example 8 Analysis of Pharmacokinetic Properties

Pharmacokinetics is concerned with the analysis of absorption,distribution, metabolism, and excretion of API. Storage and stress canpotentially affect the pharmacokinetic properties of various API. Toassess pharmacokinetics of API subject to stability and/or stresstesting, agents are incubated in containers as described in Example 6.Preferably, the API are incubated both in standard glass vials withappropriate closures and glass vials such as those provided herein. Atthe desired intervals, samples are removed and assayed.

The API is delivered to subjects by the typical route of delivery forthe API (e.g., injection, oral, topical). As pharmacokinetics areconcerned with the absorption and elimination of the API, normalsubjects are typically used to assess pharmacokinetic properties of theAPI. However, if the API is to be used in subjects with compromisedability to absorb or eliminate the API (e.g., subjects with liver orkidney disease), testing in an appropriate disease model may beadvantageous. Depending on the half-life of the compound, samples (e.g.,blood, urine, stool) are collected at predetermined time points (e.g., 0min, 30 min, 60 min, 90 min, 120 min, 4 hours, 6 hours, 12 hours, 24hours, 36 hours, 48 hours, etc.) for at least two, preferably threehalf-lives of the API, and analyzed for the presence of the API andmetabolic products of the API. At the end of the study, organs areharvested and analyzed for the presence of the API and metabolicproducts of the API.

The results are analyzed using an appropriate model selected based on,at least, the route of administration of the API. The pharmacokineticproperties of the API subjected to stability and/or stress testing arecompared to API not subjected to stability or stress testing and otherappropriate controls (e.g., vehicle control). Changes, if any, inpharmacokinetic properties as a result of storage of the API under eachcondition are determined.

Example 9 Analysis of Toxicity Profiles

Storage of API can result in alterations of toxicity of API as a resultof reactivity of the API with the container, leeching of agents from thecontainer, delamination resulting in particulates in the agent, reactionof the API molecules with each other or components of the storagebuffer, or other causes.

Agents are incubated in containers as described in the stability testingand/or stress testing methods provided in Example 6. Preferably, the APIis incubated both in standard glass vials with appropriate closures andglass vials such as those provided herein. At the desired intervals,samples are removed and assayed to determine the toxicity the API. Thetoxicity of the API is determined using methods and controls appropriateto the API. In vitro and in vivo testing can be used alone or incombination to assess changes in toxicity of agents as a result ofstorage or stress.

In in vitro assays, cell lines are grown in culture and contacted withincreasing concentrations of API subjected to stability and/or stresstesting for predetermined amounts of time (e.g., 12, 24, 36, 48, and 72hours). Cell viability is assessed using any of a number of routine orcommercially available assays. Cells are observed, for example, bymicroscopy or using fluorescence activated cell sorting (FACS) analysisusing commercially available reagents and kits. For example,membrane-permeant calcein AM is cleaved by esterases in live cells toyield cytoplasmic green fluorescence, and membrane-impermeant ethidiumhomodimer-1 labels nucleic acids of membrane-compromised cells with redfluorescence. Membrane-permeant SYTO 10 dye labels the nucleic acids oflive cells with green fluorescence, and membrane-impermeant DEAD Red dyelabels nucleic acids of membrane-compromised cells with redfluorescence. A change in the level of cell viability is detectedbetween the cells contacted with API subjected to stress and/orstability testing in standard glass vials as compared to the glass vialsprovided herein and appropriate controls (e.g., API not subject tostability testing, vehicle control).

In vivo toxicity assays are performed in animals. Typically preliminaryassays are performed on normal subjects. However, if the disease orcondition to be treated could alter the susceptibility of the subject totoxic agents (e.g., decreased liver function, decreased kidneyfunction), toxicity testing in an appropriate model of the disease orcondition can be advantageous. One or more doses of agents subjected tostability and/or stress testing are administered to animals. Typically,doses are far higher (e.g., 5 times, 10 times) the dose that would beused therapeutically and are selected, at least in part, on the toxicityof the API not subject to stability and/or stress testing. However, forthe purpose of assaying stability of API, the agent can be administeredat a single dose that is close to (e.g., 70%-90%), but not at, a dosethat would be toxic for the API not subject to stability or stresstesting. In single dose studies, after administration of the API subjectto stress and/or stability testing (e.g., 12 hours, 24 hours, 48 hours,72 hours), during which time blood, urine, and stool samples may becollected. In long term studies, animals are administered a lower dose,closer to the dose used for therapeutic treatment, and are observed forchanges indicating toxicity, e.g., weight loss, loss of appetite,physical changes, or death. In both short and long term studies, organsare harvested and analyzed to determine if the API is toxic. Organs ofmost interest are those involved in clearance of the API, e.g., liverand kidneys, and those for which toxicity would be most catastrophic,e.g., heart, brain. An analysis is performed to detect a change intoxicity between the API subjected to stress and/or stability testing instandard glass vials as compared to the glass vials provided herein, ascompared to API not subject to stability and/or stress testing andvehicle control. Changes, if any, in toxicity properties as a result ofstorage of the API under each condition are determined

Example 10 Analysis of Pharmacodynamic Profiles

Pharmacodynamics includes the study of the biochemical and physiologicaleffects of drugs on the body or on microorganisms or parasites within oron the body and the mechanisms of drug action and the relationshipbetween drug concentration and effect. Mouse models for a large varietyof disease states are known and commercially available (see, e.g.,jaxmice.jax.org/query/f?p=205:1:989373419139701::::P1_ADV:1). A numberof induced models of disease are also known.

Agents are incubated in containers as described in the stability testingand/or stress testing methods provided in Example 6. Preferably, thesamples are incubated both in standard glass vials with appropriateclosures and glass vials such as those provided herein. At the desiredintervals, samples are removed and assayed for pharmacodynamic activityusing known animal models. Exemplary mouse models for testing thevarious classes of agents indicated are known in the art.

The mouse is treated with the API subject to stability and/or stresstesting. The efficacy of the API subject to stability and/or stresstesting to treat the appropriate disease or condition is assayed ascompared to API not subject to stability and/or stress testing andvehicle control. Changes, if any, in pharmacodynamic properties as aresult of storage of the API under each condition are determined.

Example 11 Confirmation of Stability and Activity of PEDIARIX®(Diphtheria and Tetanus Toxoids and Acellular Pertussis Adsorbed,Hepatitis B (Recombinant) and Inactivated Poliovirus Vaccine)

Diphtheria and Tetanus Toxoids and Acellular Pertussis Adsorbed,Hepatitis B (Recombinant) and Inactivated Poliovirus Vaccine (PEDIARIX®)is a combination of diphtheria toxoid, tetanus toxoid, inactivatedpertussis toxin, hepatitis B virus surface antigen, inactivated Type Ipoliovirus (Mahoney), Type 2 poliovirus (MEF-1), Type 3 poliovirus(Saukett), filamentous hemagglutinin and pertactin (69 kD outer membraneprotein). Protection against disease results from the development ofneutralizing antibodies to the diphtheria toxin, the tetanus toxin, andto the poliovirus. The role of the B. pertussis components (inactivatedpertussis toxin, filamentous hemagglutinin and pertactin (69 kD outermembrane protein)) in the development of immunity to pertussis is notwell understood. Antibody concentrations >10 mIU/mL against HBsAg conferprotection against hepatitis B virus infection. The diphtheria andtetanus toxins are produced in culture and detoxified with formaldehyde.The pertussis antigens are produced in culture and the toxin isinactivated using glutaraldehye and formaldehye and the filamentoushemagglutinin and pertactin are treated with formaldehyde. The hepatitisB surface antigen is obtained from cultured cells and purified byprecipitation, ion exchange chromatography and ultrafiltration. Thethree strains of poliovirus are grown individually in cell culture,purified, inactivated using formaldehyde then pooled to form a trivalentconcentrate.

PEDIARIX® is a noninfectious, sterile vaccine for intramuscularadministration. Three 0.5 ml doses are administered at 2, 4 and 6 monthsof age. Each 0.5-ml dose contains 25 Lf of diphtheria toxoid, 10 Lf oftetanus toxoid, 25 mcg of inactivated pertussis toxin (PT), 25 mcg offilamentous hemagglutinin (FHA), 8 mcg of pertactin (69 kDa outermembrane protein), 10 mcg of HBsAg, 40 D-antigen Units (DU) of Type 1poliovirus (Mahoney), 8 DU of Type 2 poliovirus (MEF-1), and 32 DU ofType 3 poliovirus (Saukett). PEDIARIX® is a suspension for injectionavailable in 0.5 ml single-dose disposable prefilled syringe.

Diphtheria and Tetanus Toxoids and Acellular Pertussis Adsorbed,Hepatitis B (Recombinant) and Inactivated Poliovirus Vaccine samples areincubated in containers as described in the stability testing and/orstress testing methods provided in Example 6. Preferably, the samplesare incubated both in standard glass vials with appropriate closures andglass vials such as those provided herein. At the desired intervals,samples are removed and assayed to determine the stability and/oractivity of the agent. The activity of Diphtheria and Tetanus Toxoidsand Acellular Pertussis Adsorbed, Hepatitis B (Recombinant) andInactivated Poliovirus Vaccine is determined using methods and controlsappropriate to the agent, e.g., using the methods provided in Podda etal., Vaccine 9, 741-745; Gustafsson et al., N Engl J Med 334, 349-355(1996); Gillenius et al., J Biol Stand 13, 61-66 (1985); WO91/12020;WO98/00167; WO99/13906; US Publications 20110195087 and 20110206726; andU.S. Pat. No. 8,007,818; each of which is incorporated herein byreference.

In Vivo Immunogenicity Assay—Diphtheria Toxin

The in vivo immunogenicity of the diphtheria toxin is analyzed in guineapigs. A 1.0 ml dose of the vaccine subject to stability and/or stresstesting (i.e., test vaccine) is administered subcutaneously to each of48 animals. An additional 48 animals are administered 1.0 ml dose ofvaccine not subject to stability and/or stress testing (i.e., referencevaccine). The potency of the diphtheria toxin is determined by lethalchallenge method whereby 3 dilutions of each of the test and thereference vaccine are prepared. The middle dilutions contain the ED₅₀dose that saves at least, or more than, 50% of the test animals. Sixteenguinea pigs are treated for every dilution of test vaccine and referencevaccine. An additional 20 guinea pigs are not immunized and used for thetitration of the diphtheria toxin, with 5 guinea pigs used for each of 4dilutions. After 28 days the test animals are challenged with asubcutaneous dose of diphtheria toxin containing 100 LD₅₀. The samplepasses the diphtheria potency test if it contains ≧30 I.U./Single HumanDose. The test vaccine should fulfill linearity and parallelism withreference vaccine. The fiducial limit of estimated potency should bebetween 50% and 200%. The estimated potency should not be less than 30I.U. per single human dose. The 95% confidence interval of estimate ofpotency should be within 50% and 200% unless the lower limit of the 95%confidence interval of the estimated potency should be ≧30 I.U. perdose.

In Vivo Immunogenicity Assay—Tetanus Toxin

The in vivo immunogenicity of the tetanus toxin is analyzed in Swissalbino mice. A 0.5 ml dose of the vaccine subject to stability and/orstress testing (i.e., test vaccine) is administered subcutaneously toeach of 48 animals. An additional 48 animals are administered and 0.5 mldose of vaccine not subject to stability and/or stress testing (i.e.,reference vaccine). The potency of the tetanus toxin is determined bylethal challenge method whereby 3 dilutions of each of the test and thereference vaccine are prepared. The middle dilutions contain the ED₅₀dose that saves at least, or more than, 50% of the test animals. Sixteenmice are treated for every dilution of test vaccine and referencevaccine. An additional 20 mice are not immunized and used for thetitration of the tetanus toxin, with 5 mice used for each of 4dilutions. After 28 days the test animals are challenged with asubcutaneous dose of tetanus toxin containing 100 LD₅₀. The samplepasses the tetanus potency test if it contains ≧60 I.U./Single HumanDose. The test vaccine should fulfill linearity and parallelism withreference vaccine. The fiducial limit of estimated potency should bebetween 50% and 200%. The estimated potency should not be less than 60I.U. per single human dose. The 95% confidence interval of estimate ofpotency should be within 50-200% unless the lower limit of the 95%confidence interval of the estimated potency should be ≧60 I.U. perdose.

ELISA to Detect Antibody Production—Pertussis Toxin, FilamentousHemagglutinin and Pertactin

The antigenic effect of the pertussis toxin, filamentous hemagglutininand pertactin is determined in Swiss mice by an ELISA method. Threedilutions (neat, 1:5 and 1:25) of the vaccine subject to stabilityand/or stress testing (i.e., test vaccine) and vaccine not subject tostability and/or stress testing (i.e., reference vaccine) are prepared.Six groups of 8 Swiss mice are immunized using 0.5 ml of one of eachdilution by subcutaneous injection. Blood is collected 35 days afterimmunization. Serum samples are tested for antibodies against thepertussis antigens by ELISA. The test vaccine passes the potency test if≧70% of the mice receiving the test vaccine are seroconverted.

In Vivo Immunogenicity Study—Pertussis Toxin, Filamentous Hemagglutininand Pertactin

The in vivo immunogenicity of the pertussis toxin, filamentoushemagglutinin and pertactin is analyzed in twelve healthy infants whoreceive three doses of vaccine subject to stability and/or stresstesting (i.e., test vaccine) and vaccine not subject to stability and/orstress testing (i.e., reference vaccine) by intramuscular injection at2, 4 and 6 months of age. One month after the third dose of test orreference vaccine, the presence of pertussis toxin neutralizingantibodies are determined by the Chinese hamster ovary (CHO) cell assayusing U.S. reference human pertussis antiserum containing 640neutralizing units. Two-fold dilutions of 25 μl of antiserum obtainedfrom infants immunized with test or reference vaccine, and referenceantiserum, are added to culture microplate wells along with 25 μl ofpertussis toxin containing 4 times the clustering concentration andincubated for 3 hours at 37° C. Thereafter, 10,000 trypsinized CHO cellsand 200 μl of culture medium are added to each well and incubated for 48hours. Clustering is determined by direct microscopic examination and isused to measure the antibody response to the vaccine. Neutralizingtiters are expressed as the reciprocal of the highest serum dilutioncausing complete inhibition of the clustering activity induced by nativetoxin.

One month after the third dose of test or reference vaccine, levels ofIgG and IgA to the antigens is determined by ELISA. U.S. reference humanpertussis antiserum containing 200 EU/ml of IgG anti-pertussis toxin and200 EU/ml IgG anti-filamentous hemagglutinin is used as a referencestandard. An immune serum was assigned a value of 20 EU/ml of IgGanti-pertactin.

ELISA to Detect Antibody Production—Hepatitis B Surface Antigen

The antigenic effect of the hepatitis B surface antigen is determined inBalb/c mice using 5, two-fold dilutions each of vaccine subject tostability and/or stress testing (i.e., test vaccine) and vaccine notsubject to stability and/or stress testing (i.e., reference vaccine). A1.0 ml dose of test or reference vaccine is administeredintraperitoneally to 10 mice per dilution. Ten mice are inoculated withdiluent to serve as a placebo. Twenty-eight days after inoculation,blood is collected from the mice and the sera separated. The serumsamples are tested for antibody titer against hepatitis B surfaceantigen using ELISA. The test vaccine passes the hepatitis B potencytest if the upper limit of its relative potency is ≧1.

Antibody Production—Inactivated Polio Virus

The antigenic effect of the inactivated polio virus is analyzed inWistar rats using 5, three-fold dilutions each of vaccine subject tostability and/or stress testing (i.e., test vaccine) and vaccine notsubject to stability and/or stress testing (i.e., reference vaccine). A0.5 ml dose of test or reference vaccine is administered intramuscularlyto 10 rats per dilution. Twenty-one days after inoculation, blood iscollected from the rats and the serum separated. The serum samples aretested for antibody titer against type 1, type 2, and type 3 serotypesof polio virus by serum neutralization test. The test is valid if themedian effective dose (ED₅₀) for both the test and reference vaccineslies between the smallest and the largest dose given to the animals, thestatistical analysis shows no significant deviation from linearity orparallelism, and the fiducial limits of the estimated relative potencyfall between 25% and 400% of the estimated potency.

Example 12 Confirmation of Stability and Activity of HAVRIX® (HepatitisA Vaccine), ENGERIX-B® (Hepatitis B Vaccine (Recombinant)), and TWINRIX®(Hepatitis A & Hepatitis B (Recombinant) Vaccine)

Hepatitis A Vaccine (HAVRIX®) is a form of sterile suspension ofinactivated hepatitis A virus (strain HM175). The presence of antibodiesto hepatitis A virus confers protection against hepatitis A disease. Thehepatitis A virus is propagated in MRC-5 human diploid cells andinactivated with formalin. Viral antigen activity is referenced to astandard using an enzyme linked immunosorbent assay (ELISA) and isexpressed in terms of ELISA Units.

HAVRIX® is a sterile vaccine for intramuscular administration. Adultsreceive a single 1 ml dose and a 1 ml booster dose between 6 to 12months later. Children and adolescents receive a single 0.5 ml dose anda 0.5 ml booster dose between 6 to 12 months later. Each 1 ml dosecontains 1440 ELISA Units of viral antigen adsorbed onto 0.5 mg ofaluminum as aluminum hydroxide. Each 0.5 ml dose contains 720 ELISAUnits of viral antigen adsorbed onto 0.25 mg of aluminum as aluminumhydroxide. The dose also contains amino acid supplement (0.3% w/v) in aphosphate-buffered saline solution and polysorbate 20 (0.05 mg/ml).Residual compounds from the manufacturing process include not more than5 mcg/ml residual MRC-5 cellular sulfate, not more than 0.1 mg/mlformalin and not more than 40 ng/ml neomycin sulfate. HAVRIX® iscurrently supplied in either a 0.5 ml or 1 ml single-dose vial and in a0.5 ml or 1 ml prefilled syringe. HAVRIX® is a homogeneous turbid whitesuspension for injection.

Hepatitis B Vaccine (Recombinant) (ENGERIX-B®) is a form of sterilesuspension of noninfectious hepatitis B virus surface antigen (HBsAg).Protection against hepatitis B virus infection is due to antibodyconcentrations >10 mIU/mL against HBsAg. Seroconversion is defined asantibody titers ≧1 mIU/ml. The hepatitis B surface antigen is derivedfrom recombinant Saccharomyces cerevisiae cells which carry the surfaceantigen gene of the hepatitis B virus. The surface antigen is purifiedby precipitation, ion exchange chromatography and ultrafiltration.

ENGERIX-B® is a sterile vaccine for intramuscular administration.Individuals 20 years of age and older receive a series of 3, 1 ml dosesgiven on a 0, 1, and 6 month schedule. Individuals from birth to 19years of age receive a series of 3, 0.5 ml doses given on a 0, 1, and 6month schedule. Each 1 ml dose contains 20 mcg of HBsAg adsorbed on 0.5mg aluminum as aluminum hydroxide. Each 0.5 ml dose contains 10 mcg ofHBsAg adsorbed on 0.25 mg aluminum as aluminum hydroxide. Excipientsinclude sodium chloride (9 mg/ml) and phosphate buffers (disodiumphosphate dihydrate, 0.98 mg/ml; sodium dihydrogen phosphate dihydrate,0.71 mg/ml). Each dose contains no more than 5% yeast protein. ENGERIX®is currently supplied in either a 0.5 ml or 1 ml single-dose vial and ina 0.5 ml or 1 ml prefilled syringe. ENGERIX® is a homogeneous turbidwhite suspension for injection.

Hepatitis A & Hepatitis B (Recombinant) Vaccine (TWINRIX®) is a bivalentvaccine containing inactivated hepatitis A virus (strain HM175) andnoninfectious hepatitis B virus surface antigen (HBsAg). The presence ofantibodies to hepatitis A virus confers protection against hepatitis Adisease. Protection against hepatitis B virus infection is due toantibody concentrations >10 mIU/mL against HBsAg. The hepatitis A virusis propagated in cell culture and inactivated with formalin. Thehepatitis B surface antigen is obtained from cultured cells and purifiedby precipitation, ion exchange chromatography and ultrafiltration.

TWINRIX® is a sterile vaccine for intramuscular administration.Individuals receive a standard dosing regimen of 3, 1 ml doses at 0, 1,and 6 months. Under an accelerated dosing regimen, individuals receive4, 1 ml doses on days 0, 7, and 21 to 30 followed by a booster dose at12 months. Each 1 ml dose contains 720 ELISA Units of inactivatedhepatitis A virus and 20 mcg of recombinant HBsAg protein. The 1 ml doseof vaccine also contains 0.45 mg of aluminum in the form of aluminumphosphate and aluminum hydroxide as adjuvants, amino acids, sodiumchloride, phosphate buffer, polysorbate 20 and Water for Injection.TWINRIX® is currently supplied as a 1 ml single-dose vial and as aprefilled syringe and is formulated as a turbid white suspension forinjection.

Hepatitis A Vaccine, Hepatitis B Vaccine (Recombinant), and aformulation including both vaccines, are incubated in containers asdescribed in the stability testing and/or stress testing methodsprovided in Example 6. Preferably, the samples are incubated both instandard glass vials with appropriate closures and glass vials such asthose provided herein. At the desired intervals, samples are removed andassayed to determine the stability and/or activity of the agent. Theactivity of Hepatitis A Vaccine, Hepatitis B Vaccine (Recombinant), anda formulation including both vaccines, is determined using methods andcontrols appropriate to the agent, e.g., using the methods provided inEP1073462B1; US Publication 20110195087; U.S. Pat. No. 5,151,023; andU.S. Pat. No. 7,144,703, each of which is incorporated herein byreference.

ELISA to Detect Antibody Production—Hepatitis B Surface Antigen

The antigenic effect of the hepatitis B surface antigen is determined inBalb/c mice using 5, two-fold dilutions each of vaccine subject tostability and/or stress testing (i.e., test vaccine) and vaccine notsubject to stability and/or stress testing (i.e., reference vaccine). A1.0 ml dose of test or reference vaccine is administeredintraperitoneally to 10 mice per dilution. Ten mice are inoculated withdiluent to serve as a placebo. Twenty-eight days after inoculation,blood is collected from the mice and the sera separated. The serumsamples are tested for antibody titer against hepatitis B surfaceantigen using ELISA. The test vaccine passes the hepatitis B potencytest if the upper limit of its relative potency is ≧1.

ELISA to Detect Antibody Production—Hepatitis a Virus Antigen andHepatitis B Surface Antigen

The antigenic effect of the hepatitis A virus antigen and hepatitis Bsurface antigen is determined in 4 week old SPF guinea pigssubcutaneously immunized with 200 μl of the vaccine. Vaccine comprisingonly the hepatitis A viral antigen, only the hepatitis B surface antigenor a combination of both may be analyzed by this method. Ten animals areimmunized with the vaccine subject to stability and/or stress testing(i.e., test vaccine) and 10 animals are immunized with vaccine notsubject to stability and/or stress testing (i.e., reference vaccine).Six weeks after immunization blood is collected and the sera separated.The serum samples are tested for antibody titers against hepatitis Avirus antigen and/or hepatitis B surface antigen using ELISA.

The anti-hepatitis A viral antibody titer is determined using acompetitive inhibitory ELISA technique. Specifically, a microplate wellis coated with anti-hepatitis A virus rabbit serum and blocked with BSAthen reacted with hepatitis A viral antigen at 4° C. overnight.Thereafter, serum obtained from test or reference vaccine immunizedanimals is added to the well and incubated at room temperature for 30minutes. Following this, a peroxidase-labeled anti-hepatitis A virusrabbit antibody is added to the well and incubated at 37° C. for 2hours. The substrate solution is added for color development. Thereaction is stopped and the absorbance at 492 nm is measured and theantigen titer calculated as a titer at which the inhibition rate is 50%based on the calibration curve of a standard material. The antibody usedas a standard material is prepared so that it demonstrates a relativetiter of 2 IU/ml when the anti-hepatitis A virus antibody titer of theAnti-Hepatitis A Reference Globin No. 1 from the Bureau of Biologics ofthe F.D.A is set at 100 IU/ml.

The anti-hepatitis B surface antigen antibody titer is determined usingan AUSAB kit (Abbott Co., Ltd) and based on a calibration curveddeveloped using the WHO International Reference (I-HGIB, 50 IU/ml).

Example 13 Confirmation of Stability and Activity of EPERZAN®(Albiglutide)

Albiglutide (EPERZAN®) is a glucagon-like peptide-1 (GLP-1) receptoragonist. GLP-1 is secreted by the gastrointestinal tract during eatingand increases insulin secretion, decreases glycemic excursion, delaysgastric emptying and reduces food intake. Albiglutide is presentlyindicated for treatment of adults with type 2 diabetes.

Albiglutide is a GLP-1 receptor agonist comprising a dipeptidylpeptidase-4 resistant GLP-1 dimer fused in series to human albuminNative GLP-1 peptide is rapidly degraded while albiglutide has a longerduration of action (e.g. half-life of 4 to 7 days) due to its resistanceto hydrolysis by dipeptidyl peptidase-4. Albiglutide is designed forweekly or biweekly subcutaneous dosing. Albiglutide may be provided at aconcentration of 50 mg/mL following resuspension of a lyophilized formcomprising 2.8% mannitol, 4.2% trehalose dihydrate, 0.01% polysorbate80, 10 to 20 mM phosphate buffer at pH 7.2. The formulation is dilutedwith water for injection as necessary for respective dosing.

Albiglutide samples are incubated in containers as described in thestability testing and/or stress testing methods provided in Example 6.Preferably, the samples are incubated both in standard glass vials withappropriate closures and glass vials such as those provided herein. Atthe desired intervals, samples are removed and assayed to determine theactivity of the agent in, at least, on in vitro or in vivo assay toassess the biological activity of albiglutide. The activity ofalbiglutide is determined using methods and controls appropriate to theagent, for example using methods provided in Baggio et al., Diabetes 53,2492-2500 (2004); US Publication 20110301080 A1; or any one of U.S. Pat.Nos. 7,141,547; 7,238,667; 7,592,010; 7,799,759; 7,847,079; 8,012,464;8,071,539; 8,211,439; and 8,252,739, each of which is incorporatedherein by reference.

Measurement of cAMP to Detect Activation of GLP-1 Receptor

The ability of albiglutide subject to stability and/or stress testing(i.e., test compound) or albiglutide not subject to stability and/orstress testing (i.e., reference compound) to activate the GLP-1 receptorin vitro is determined by measuring the accumulation of cAMP in babyhamster kidney cells stably transfected with rat GLP-1 receptor(BHK-GLP-1R). BHK-GLP-1R cells are grown to 70-80% confluence in 24-wellplates in the absence of G418 at 37° C. Cells are incubated withDulbecco's modified Eagle's medium containing serum and 100 μmol/L3-isobutyl-1-methylxanthine for 5 minutes at 37° C., followed by anadditional 10 minute incubation in the presence of increasingconcentrations of test or reference compound. All reactions are carriedout in triplicate and terminated by addition of ice-cold absoluteethanol. Cell extracts are lyophilized and cAMP levels measured using acAMP radioimmunoassay kit (Biomedical Technologies, Stoughton Mass.).

In Vivo Activation of GLP-1 Receptor

To determine whether albiglutide subject to stability and/or stresstesting or albiglutide not subject to stability and/or stress testing iscapable of reaching body sites and activating GLP-1 receptor dependentprocesses in vivo, glucose tolerance tests, measurement of plasmainsulin, feeding studies, gastric emptying studies, and c-FOS activationstudies are conducted.

Glucose Tolerance Tests and Measurement of Plasma Insulin

The ability of albiglutide subject to stability and/or stress testing(i.e., test compound) or albiglutide not subject to stability and/orstress testing (i.e., reference compound) to modulate blood glucose andplasma insulin levels is measured in C57BL/6 mice. Following anovernight (16-18 hour) fast, mice are treated with test or referencecompound followed by administration of 1.5 mg/g body weight of glucoseorally or by injection into the peritoneal cavity. A blood sample iscollected from the tail vein at 0, 10, 20, 30, 60, 90 and 120 minutesafter glucose administration. Blood glucose concentrations are measuredusing a Glucometer Elite blood glucose meter (Bayer, Toronto, Ontario,Canada). Plasma insulin is measured in a 100 μl blood sample collectedfrom the tail vein during the 10 and 20 minute time periods afterglucose administration. The blood sample is immediately mixed with 10%volume of a chilled solution containing 5,000 KIU/ml Trasylol (Bayer),32 mmol/l EDTA, and 0.1 nmol/l diprotin A. Plasma insulin is measuredusing a rat insulin enzyme-linked immunosorbent assay kit (Crystal Chem,Chicago Ill.) with mouse insulin as a standard.

Feeding Studies

The ability of albiglutide subject to stability and/or stress testing(i.e., test compound) or albiglutide not subject to stability and/orstress testing (i.e., reference compound) to inhibit food intake ismeasured in C57BL/6 mice. After an overnight fast (16 hours), mice areinjected intracerebroventrically or intraperitoneally with control (PBSor human serum albumin), or test or reference compound. Mice receiving a5 μl injection into the lateral ventricle are allowed to recover fromanesthesia, then assessed for food intake. Mice that receive a 100 μlintraperitoneal injection are weighed and then placed into individualcages containing preweighed rodent food and water. At 2, 4, 7, and 24hours after reagent administration, the food is reweighed and total foodintake calculated.

Gastric Emptying Studies

The ability of albiglutide subject to stability and/or stress testing(i.e., test compound) or albiglutide not subject to stability and/orstress testing (i.e., reference compound) to inhibit gastric emptying ismeasured in C57BL/6 mice. After an overnight fast (18 hours), mice areallowed free access to preweighed food for 1 hour. After the 1 hourre-feeding, the remaining food is weighed and food intake is determined.Mice are injected intraperitoneally with PBS, human serum albumin (2.7mg/kg), or test or reference compound (3 mg/kg) then deprived of foodfor an additional 4 hours. Following this, the mice are anesthetized andtheir stomachs removed. The stomach content wet weight is determinedGastric emptying rate is calculated using the following: gastricemptying rate (%)=[1−(stomach content wet weight/food intake)]×100.

C-Fos Activation Studies

The ability of albiglutide subject to stability and/or stress testing(i.e., test compound) or astuprotimut-R not subject to stability and/orstress testing (i.e., reference compound) to activate c-FOS expressionis measured in C57BL/6 mice brains. Mice are given intraperitonealinjections of PBS, human serum albumin, albiglutide subject to stabilityand/or stress testing, or albiglutide not subject to stability and/orstress testing in a 100 μl volume. At 10 and 60 minutes after injection,mice are anesthetized and perfused intracardially with ice-cold normalsaline followed by 4% paraformaldehyde solution. Brains are removedimmediately at the end of the perfusion, kept cold in ice cold 4%paraformaldehyde solution for 3 days then transferred to a solutioncontaining paraformaldehyde and 10% sucrose for 12 hours. Brains are cutinto 25 μm sections using a Lecia SM2000R sliding microtome and storedat −20° C. in a cold cryoprotecting solution. Sections are processed forimmunocytochemical detection of c-FOS using anavidin-biotin-immunoperoxidase method. The c-FOS antibody is used at a1:50,000 dilution. Brain sections corresponding to the level of the areapostrema, the nucleus of the solitary tract, the central nucleus of theamygdala and the parabrachial and paraventricular nuclei are selectedfor quantitative assessment of c-FOS immunoreactive neurons.

Example 14 Confirmation of Stability and Activity of MAGE-A3Antigen-Specific Cancer Immunnotherapeutic (Astuprotimut-R)

Astuprotimut-R (MAGE-A3 Antigen-Specific Cancer Immunotherapeutic) is aform of fusion protein consisting of MAGE-A3 (melanoma associatedantigen 3) epitope. MAGE-A3 may also be referred to as MAGE-3. A MAGE-A3fusion protein may induce a therapeutic effect by stimulating acytotoxic T lymphocyte (CTL) response against tumor cells that expressthe MAGE-A3 antigen, resulting in tumor cell death. Astuprotimut-R is afusion protein consisting of the MAGE-A3 (melanoma associated antigen 3)epitope fused to part of the Haemophilus influenza protein D antigensequence. MAGE-A3 is a tumor specific antigen expressed in a variety ofcancers (e.g., melanoma, non-small cell lung cancer, head and neckcancer and bladder cancer). Astuprotimut-R is administered along with anadjuvant system comprising specific combinations of immunostimulatingcompounds selected to increase the anti-tumor response.

Astuprotimut-R samples are incubated in containers as described in thestability testing and/or stress testing methods provided in Example 6.Preferably, the samples are incubated both in standard glass vials withappropriate closures and glass vials such as those provided herein. Atthe desired intervals, samples are removed and assayed to determine theactivity of the agent in, at least, on in vitro or in vivo assay toassess the biological activity of albiglutide. The activity ofastuprotimut-R is determined using methods and controls appropriate tothe agent, for example using methods provided in Vantomme et al., JImmunother 27, 124-135 (2004); WO99/40188; U.S. Pat. No. 7,049,413; U.S.Pat. No. 7,371,845; U.S. Pat. No. 7,388,072; and US Publication20090203124, each of which is incorporated herein by reference.

ELISA Assay to Detect Antibody Production

The antigenic effect of astuprotimut-R is determined in 2 differentmouse strains (C57BL/6 and Balb/C). Five mice of each strain areinjected twice at 2 week intervals in the foot pad with 5 μg ofastuprotimut-R subject to stability and/or stress testing (i.e., testvaccine) or astuprotimut-R not subject to stability and/or stresstesting (i.e., reference vaccine) at 1/10th of the concentration used inhuman settings.

Sera are prepared from blood collected from the mice 2 weeks after thelast injection. Two μg/ml of purified antigen is used as coated antigen.After saturation at 1 hour at 37° C., in PBS with 1% newborn calf serum,the sera are serially diluted (starting at 1/1000) and incubatedovernight at 4° C., or 90 minutes at 37° C. After washing in PBS/Tween20, biotinylated goat anti-mouse total IgG (1/1000) or goat anti-mouseIgG1, IgG2a, IgG2b antisera (1/5000) are used as second antibodies.After 90 minutes incubation at 37° C., streptavidin coupled toperoxidase is added, and TMB (tetra-methyl-benzidine peroxide) is usedas substrate. After 10 minutes the reaction is blocked by addition ofH₂SO₄ 0.5M, and the O.D. is determined.

Proliferation Assay to Detect Immune Response

The immunogenic effect of astuprotimut-R is determined in 2 differentmouse strains (C57BL/6 and Balb/C). Five mice of each strain areinjected twice at 2 week intervals in the foot pad with 5 μg ofastuprotimut-R subject to stability and/or stress testing (i.e., testvaccine) or astuprotimut-R not subject to stability and/or stresstesting (i.e., reference vaccine) at 1/10th of the concentration used inhuman settings.

Lymphocytes for proliferation assays are prepared by sacrificing themice followed by collection and crushing of the spleen or popliteallymph nodes 2 weeks after the last injection. 2×10⁵ cells are placed intriplicate in 96 well plates and the cells are re-stimulated in vitrofor 72 hours with different concentrations (1-0.1 μg/ml) of antigen orantigen coated onto latex micro-beads. The lymphoproliferative responseof mice receiving either test or reference vaccine is documented.

Gel Electrophoresis Methods for Size Detection

Samples of astuprotimut-R subject to stability and/or stress testing ornot subject to stability and/or stress testing are diluted and resolvedby gel electrophoresis. SDS-PAGE is used to determine protein size andaggregate formation. Native gel electrophoresis is used to analyzeprotein complexes formed by covalent or non-covalent interaction.Non-reducing gel electrophoresis is used to analyze protein complexes orprotein structures containing disulfide bonds. Proteins resolved by gelelectrophoresis are detected using sensitive staining methods, such assilver stain. Gel electrophoresis and silver staining methods are knownin the art.

Example 15 Confirmation of Stability and Activity of GSK2402968(Drisapersen)

GSK2402968 (drisapersen) is a dystrophin antisense oligonucleotide, oran analog thereof. Antisense oligonucleotides may affect thepost-transcriptional splicing process of premRNA to restore the readingframe of the mRNA, resulting in a shortened dystrophin protein.Drisapersen is currently indicated for the treatment of boys withDuchenne Muscular Dystrophy with a dystrophin gene mutation amenable toan exon 51 skip. Drisapersen is an antisense oligonucleotide(5′-UCAAGGAAGAUGGCAUUUCA-3′) (SEQ ID NO: 1) with full length2′-O-methyl-substituted ribose moieties and phosphorothioateinternucleotide linkages. Binding of the antisense oligonucleotide toeither one or both splice sites or exon-internal sequences inducesskipping of the exon by blocking recognition of the splice sites by thespliceosome complex. Drisapersen may be provided in 0.5 ml glass vialsin sodium phosphate buffered saline at a concentration of 100 mg/ml. Itmay be formulated for abdominal subcutaneous administration at a dose of0.5 to 10 mg per kg of body weight.

To determine the stability of drisapersen, drisapersen samples areincubated in containers as described in the stability testing and/orstress testing methods provided in Example 6. Preferably, the samplesare incubated both in standard glass vials with appropriate closures andglass vials such as those provided herein. At the desired intervals,samples are removed and assayed to determine the stability and/oractivity of the agent. The stability and/or activity of drisapersen isdetermined using methods and controls appropriate to the agent, forexample, using methods provided in Aartsma-Rus et al., Hum Molec Genet.12, 907-914 (2003); van Deutekom et al., N Engl J Med 357, 2677-2686;Goemans et al., N Engl J Med 364, 1513-1522 (2011); US PatentPublications US 20080209581, US20090228998, US20110294753 andUS20130035367; U.S. Pat. No. 7,973,015; and U.S. Pat. No. 8,304,398,each of which is incorporated herein by reference.

Gel Mobility Shift Assays to Detect Binding Affinity

The binding affinity of drisapersen subject to stability and/or stresstesting (i.e., test oligo) or drisapersen not subject to stabilityand/or stress testing (i.e., reference oligo) for the target sequence isdemonstrated using a gel mobility shift assay. The exon 51 target RNAfragment is generated by in vitro T7-transcription from a PCR fragment(amplified from either murine or human muscle mRNA using a sense primerthat contains the T7 promoter sequence) in the presence of ³²P-CTP. Thebinding affinity of test or reference oligo (0.5 pmol) for the targettranscript fragments is determined by hybridization at 37° C. for 30minutes and subsequent polyacrylamide (8%) gel electrophoresis.Drisapersen generates a mobility shift, demonstrating its bindingaffinity for the target RNA.

RT-PCR and Sequence Analysis to Detect Exon Skipping in Muscle CellCultures

The ability of drisapersen subject to stability and/or stress testing(i.e., test oligo) or drisapersen not subject to stability and/or stresstesting (i.e., reference oligo) to induce skipping in muscle cells invitro is measured in proliferating mouse myoblasts and post-mitoticmouse myotube cultures (expressing higher levels of dystrophin) derivedfrom the mouse muscle cell line C2C12. The analysis is also conducted inhuman-derived primary muscle cell cultures isolated from muscle biopsiesfrom Duchenne Muscular Dystrophy patients carrying a dystrophin genemutation amenable to an exon 51 skip. These heterogeneous culturescontain approximately 20-40% myogenic cells. The test or reference oligo(at a concentration of 1 μM) is transfected into the cells using thecationic polymer PEI (MBI Fermentas) at a ratio-equivalent of 3. Thetest or reference oligo used in these experiments contains a 5′fluorescein group which allows for the determination of the transfectionefficiencies by counting the number of fluorescent nuclei. Typically,more than 60% of cells show specific nuclear uptake of the drisapersen.

Following transfection, RNA is isolated 24 hours later using RNAzol B(CamPro Scientific, The Netherlands). RNA is reverse transcribed usingC. therm polymerase (Roche) and either a mouse or a human dystrophinspecific reverse primer. To facilitate the detection of skipping ofdystrophin exon 51, the cDNA is amplified by two rounds of PCR,including a nested amplification using either mouse or human dystrophinspecific primers. A truncated product corresponding to the RNA caused byexon 51 skipping is detected. Subsequent sequence analysis confirmsspecific skipping of exons in the dystrophin transcripts.

Immunohistochemical Analysis to Detect Dystrophin in Human Muscle CellCultures

The ability of drisapersen subject to stability and/or stress testing(i.e., test oligo) or drisapersen not subject to stability and/or stresstesting (i.e., reference oligo) to induce the skipping of exon 51 inmuscles cells from patients carrying deletions amenable to an exon 51skip and to restore the translation and synthesis of a dystrophinprotein is shown using immunohistochemical analysis. Patient derivedmuscle cell culture are transfected with test or reference oligo thensubjected to immunocytochemistry using two different dystrophinmonoclonal antibodies raised against domains of the dystrophin proteinlocated proximal and distal of the targeted region respectively.Fluorescent analysis reveals restoration of dystrophin synthesis inpatient-derived cell cultures. Approximately at least 80% of the fiberswill stain positive for dystrophin in the treated samples.

Example 16 Confirmation of Stability and Activity of HZ/Su (HerpesZoster Virus Vaccine)

Herpes zoster virus vaccine (HZ/su) is a form of recombinant varicellazoster virus glycoprotein E. The varicella zoster virus glycoprotein Esubunit is the most abundant glycoprotein in the varicella zoster virusand induces a potent CD4+ T-cell response when administered with anadjuvant. Herpes zoster virus vaccine is formulated for intramuscularinjection and comprises 50 μg of varicella zoster virus glycoprotein Ein 0.2 ml mixed with 0.5 ml of AS01B adjuvant. AS01B adjuvant is aliposome based adjuvant system containing 50 μg3-O-desacyl-4′-monophosphoryl lipid A (MPL) and 50 μg QS21 (a triterpeneglycoside).

To determine the stability of herpes zoster virus vaccine, herpes zostervirus vaccine samples are incubated in containers as described in thestability testing and/or stress testing methods provided in Example 6.Preferably, the samples are incubated both in standard glass vials withappropriate closures and glass vials such as those provided herein. Atthe desired intervals, samples are removed and assayed to determine thestability and/or activity of the agent. The stability and/or activity ofherpes zoster virus vaccine is determined using methods and controlsappropriate to the agent, for example, using methods provided in any ofJacquet, et al., Vaccine 20, 1593-1602 (2002); Leroux-Roels, et al., JInfect Dis 15, 1280-1290 (2012); Haumont, et al., J Med Virol 53, 63-68(1997); ans WO2006/094756, each of which is incorporated herein byreference.

ELISA to Detect Antibody Production

The antigenic effect of HZ/su is determined in guinea pigs and mice.Three groups of 6 female Durkin Hartlay guinea pigs are subcutaneouslyimmunized twice at 28 day intervals with 8 μg of HZ/su subject tostability and/or stress testing (i.e., test vaccine), 8 μg of HZ/su notsubject to stability and/or stress testing (i.e., reference vaccine), oradjuvant alone. Blood samples are collected on days 28, 42 and 72. Threegroups of 6 female BalbC mice are subcutaneously immunized twice at 28day intervals with 5 n of HZ/su subject to stability and/or stresstesting (i.e., test vaccine), 5 μg of HZ/su not subject to stabilityand/or stress testing (i.e., reference vaccine), or adjuvant alone.Blood samples are collected on days 28 and 42. Mice are sacrificed onday 42 and their spleens collected.

Sera prepared from the blood samples is analyzed for anti-recgEantibodies by ELISA. Immunoplates are coated with recgE at 4° C. Platesare washed 5 times with TBS-Tween (50 mM Tris-HCl pH 7.5, 150 mM NaCl,0.1% Tween 80) and saturated for 1 hour at 37° C. with 150 μl of thesame buffer supplemented with 1% BSA. Serial dilutions of sera insaturation buffer are incubated for 1 hour at 37° C. Plates are washed 5times with TBS-Tween buffer and antigen-bound antibodies are detectedwith the second antibody (goat anti-mouse or goat anti-guinea pig IgG)coupled to alkaline phosphatase (dilution 1/7500 in TBS-Tween buffer).The enzymatic activity is measured using the p-nitrophenylphosphatesubstrate dissolved in diethanolamine buffer (pH 9.8). OD 415 nm ismeasured in an ELISA reader.

Mouse antibody subclass is determined using immunoplates coated asdescribed above and IgG1- or IgG2a-specific biotin-labeled monoclonalantibodies (rat anti-mouse, dilution 1/7000 in TBS-Tween buffer and 1%BSA) as second antibodies. Phosphatase alkaline-conjugated streptavidin(1/1000 dilution) is added to each well. The enzymatic activity ismeasured using the p-nitrophenylphosphate substrate dissolved indiethanolamine buffer (pH 9.8). OD 415 nm is measured in an ELISAreader.

ELISA titers are identified as the reciprocal of the dilution giving asignal corresponding to 50% of the maximal OD 415 nm value.

Neutralization Assay to Detect Antibody Mediated Neutralization ofVaricella Zoster Virus Infectivity

The ability of HZ/su to stimulate the production of neutralizingantibodies is analyzed using sera collected from mice treated asdescribed above. Varicella zoster virus (100 μl at 4×10² pfu/ml) isincubated for 1 hour at 37° C. with 100 μl of heat-inactivated mouseserum serially diluted in buffer (PBS, saccharose 5%, glutamate 1%,fetal bovine serum 10%, pH 7.1). Guinea pig serum complement (2 μl) isadded to each well and incubated for 2 hours at 37° C. then plated ontoa confluent MRC-5 monolayer at room temperature in the dark. Two hourslater, 800 μl of culture medium (RPMI plus 2% fetal bovine serum) isadded. Seven days later, the medium is removed, and cells are fixed andstained with Coomassie blue solution for 10 minutes. Plates are washedwith distilled water and lysis plaques are counted. Each dilution istested in duplicate. The neutralizing titer is expressed as thereciprocal of the serum dilution resulting in 50% neutralization.

It will be apparent to those skilled in the art that variousmodifications and variations can be made to the embodiments describedherein without departing from the spirit and scope of the claimedsubject matter. Thus it is intended that the specification cover themodifications and variations of the various embodiments described hereinprovided such modification and variations come within the scope of theappended claims and their equivalents.

1-17. (canceled)
 18. A pharmaceutical composition comprising: PEDIARIX®(Diphtheria and Tetanus Toxoids and Acellular Pertussis Adsorbed,Hepatitis B (Recombinant) and Inactivated Poliovirus Vaccine), HAVRIX®(Hepatitis A Vaccine), ENGERIX-B® (Hepatitis B Vaccine (Recombinant)),TWINRIX® (Hepatitis A & Hepatitis B (Recombinant) Vaccine), EPERZAN®(albiglutide), MAGE-A3 Antigen-Specific Cancer Immunotherapeutic(astuprotimut-R), GSK2402968 (drisapersen), or HZ/su (herpes zostervaccine) and a pharmaceutically acceptable excipient; wherein thepharmaceutical composition is contained within a glass pharmaceuticalcontainer comprising a glass composition comprising: SiO₂ in a an amountgreater than or equal to about 72 mol. % and less than or equal to about78 mol. %; alkaline earth oxide comprising both MgO and CaO, wherein CaOis present in an amount up to about 1.0 mol. %, and a ratio (CaO (mol.%)/(CaO (mol. %)+MgO (mol. %))) is less than or equal to 0.5; X mol. %Al₂O₃, wherein X is greater than or equal to about 5 mol. % and lessthan or equal to about 7 mol. %; Y mol. % alkali oxide, wherein thealkali oxide comprises Na₂O in an amount greater than about 8 mol. %;and a ratio of a concentration of B₂O₃ (mol. %) in the glass containerto (Y mol. %−X mol. %) is less than or equal to 0.3.
 19. Thepharmaceutical composition of claim 18, wherein the pharmaceuticalcontainer comprises a compressive stress greater than or equal to 150MPa.
 20. The pharmaceutical composition of claim 18, wherein thepharmaceutical container comprises a compressive stress greater than orequal to 250 MPa.
 21. The pharmaceutical composition of claim 18,wherein the pharmaceutical container comprises a depth of layer greaterthan 30 μm.
 22. (canceled)
 23. The pharmaceutical composition of claim18, wherein the pharmaceutical composition comprises increasedstability, product integrity, or efficacy.
 24. A pharmaceuticalcomposition comprising: PEDIARIX® (Diphtheria and Tetanus Toxoids andAcellular Pertussis Adsorbed, Hepatitis B (Recombinant) and InactivatedPoliovirus Vaccine), HAVRIX® (Hepatitis A Vaccine), ENGERIX-B®(Hepatitis B Vaccine (Recombinant)), TWINRIX® (Hepatitis A & Hepatitis B(Recombinant) Vaccine), EPERZAN® (albiglutide), MAGE-A3 Antigen-SpecificCancer Immunotherapeutic (astuprotimut-R), GSK2402968 (drisapersen), orHZ/su (herpes zoster vaccine) and a pharmaceutically acceptableexcipient; wherein the pharmaceutical composition is contained within aglass pharmaceutical container comprising an internal homogeneous layerand a compressive stress greater than or equal to 150 MPa.
 25. Thepharmaceutical composition of claim 24, wherein the pharmaceuticalcontainer comprises a depth of layer greater than 10 μm.
 26. Thepharmaceutical composition of claim 25, wherein the pharmaceuticalcontainer comprises a depth of layer greater than 25 μm.
 27. Thepharmaceutical composition of claim 24, wherein the pharmaceuticalcontainer has a delamination factor of less than
 3. 28. (canceled) 29.The pharmaceutical composition of claim 24, wherein the pharmaceuticalcontainer comprises increased stability, product integrity, or efficacy.30. A pharmaceutical composition comprising: PEDIARIX® (Diphtheria andTetanus Toxoids and Acellular Pertussis Adsorbed, Hepatitis B(Recombinant) and Inactivated Poliovirus Vaccine), HAVRIX® (Hepatitis AVaccine), ENGERIX-B® (Hepatitis B Vaccine (Recombinant)), TWINRIX®(Hepatitis A & Hepatitis B (Recombinant) Vaccine), EPERZAN®(albiglutide), MAGE-A3 Antigen-Specific Cancer Immunotherapeutic(astuprotimut-R), GSK2402968 (drisapersen), or HZ/su (herpes zostervaccine) and a pharmaceutically acceptable excipient; wherein thepharmaceutical composition is contained within a glass pharmaceuticalcontainer having a compressive stress greater than or equal to 150 MPaand a depth of layer greater than 10 μm, and wherein the pharmaceuticalcomposition comprises increased stability, product integrity, orefficacy.
 31. (canceled)
 32. A pharmaceutical composition comprising:PEDIARIX® (Diphtheria and Tetanus Toxoids and Acellular PertussisAdsorbed, Hepatitis B (Recombinant) and Inactivated Poliovirus Vaccine),HAVRIX® (Hepatitis A Vaccine), ENGERIX-B® (Hepatitis B Vaccine(Recombinant)), TWINRIX® (Hepatitis A & Hepatitis B (Recombinant)Vaccine), EPERZAN® (albiglutide), MAGE-A3 Antigen-Specific CancerImmunotherapeutic (astuprotimut-R), GSK2402968 (drisapersen), or HZ/su(herpes zoster vaccine) and a pharmaceutically acceptable excipient;wherein the pharmaceutical composition is contained within a glasspharmaceutical container comprising a delamination factor of less than3, wherein the pharmaceutical composition comprises increased stability,product integrity, or efficacy.
 33. A pharmaceutical compositioncomprising: PEDIARIX® (Diphtheria and Tetanus Toxoids and AcellularPertussis Adsorbed, Hepatitis B (Recombinant) and Inactivated PoliovirusVaccine), HAVRIX® (Hepatitis A Vaccine), ENGERIX-B® (Hepatitis B Vaccine(Recombinant)), TWINRIX® (Hepatitis A & Hepatitis B (Recombinant)Vaccine), EPERZAN® (albiglutide), MAGE-A3 Antigen-Specific CancerImmunotherapeutic (astuprotimut-R), GSK2402968 (drisapersen), or HZ/su(herpes zoster vaccine) and a pharmaceutically acceptable excipient;wherein the pharmaceutical composition is contained within a glasspharmaceutical container which is substantially free of boron, andwherein the pharmaceutical composition comprises increased stability,product integrity, or efficacy.
 34. The pharmaceutical composition ofclaim 33, wherein the glass pharmaceutical container comprises acompressive stress greater than or equal to 150 MPa and a depth of layergreater than 25 μm.
 35. The pharmaceutical composition of claim 34,wherein the glass pharmaceutical container comprises a compressivestress greater than or equal to 300 MPa and a depth of layer greaterthan 35 μm.
 36. The pharmaceutical composition of claim 33, wherein saidglass pharmaceutical container comprises a substantially homogeneousinner layer.
 37. The pharmaceutical composition of claim 36, whereinsaid glass pharmaceutical container comprises a compressive stressgreater than or equal to 150 MPa and a depth of layer greater than 25μm. 38-44. (canceled)
 45. The pharmaceutical composition of claim 18,wherein the pharmaceutical container comprises an internal homogeneouslayer.
 46. The pharmaceutical composition of claim 27, wherein thepharmaceutical container comprises increased stability, productintegrity, or efficacy.